U.S. patent application number 09/309196 was filed with the patent office on 2003-01-09 for yeast cells engineered to produce pheromone system protein surrogates, and uses therefor.
Invention is credited to BROACH, JIM, FOWLKES, DANA MERRIMAN, KLEIN, CHRISTINE, MANFREDI, JOHN, MURPHY, ANDREW J., PAUL, DR. JEREMY, TRUEHEART, JOSHUA.
Application Number | 20030008380 09/309196 |
Document ID | / |
Family ID | 27488722 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008380 |
Kind Code |
A1 |
FOWLKES, DANA MERRIMAN ; et
al. |
January 9, 2003 |
Yeast cells engineered to produce pheromone system protein
surrogates, and uses therefor
Abstract
Yeast cells are engineered to express both a surrogate of a
pheromone system protein (e.g., enzymes involved in maturation of
.alpha.-factor, transporters of a-factor, pheromone receptors,
etc.) and a potential peptide modulator of the surrogate, in such a
manner that the inhibition or activation of the surrogate affects a
screenable or selectable trait of the yeast cells. Various
additional features improve the signal-to-noise ratio of the
screening/selection system.
Inventors: |
FOWLKES, DANA MERRIMAN;
(CHAPEL HILL, NC) ; BROACH, JIM; (PRINCETON,
NJ) ; MANFREDI, JOHN; (NEW YORK, NY) ; KLEIN,
CHRISTINE; (NEW YORK, NY) ; MURPHY, ANDREW J.;
(MONTCLAIR, NJ) ; PAUL, DR. JEREMY; (SOUTH NYACK,
NY) ; TRUEHEART, JOSHUA; (SOUTH NYACK, NY) |
Correspondence
Address: |
GIULIO A. DECONTI, JR.
LAHIVE & COCKFIELD, LLP
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
27488722 |
Appl. No.: |
09/309196 |
Filed: |
May 10, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09309196 |
May 10, 1999 |
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08322137 |
Oct 13, 1994 |
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6100042 |
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08322137 |
Oct 13, 1994 |
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08309313 |
Sep 20, 1994 |
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08309313 |
Sep 20, 1994 |
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08190328 |
Jan 31, 1994 |
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08190328 |
Jan 31, 1994 |
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08041431 |
Mar 31, 1993 |
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Current U.S.
Class: |
435/254.2 ;
435/7.31 |
Current CPC
Class: |
C07K 2319/02 20130101;
C12N 15/81 20130101; C07K 14/395 20130101 |
Class at
Publication: |
435/254.2 ;
435/7.31 |
International
Class: |
G01N 033/53; G01N
033/569; C12N 001/14; C12N 001/16; C12N 001/18 |
Claims
1. A yeast cell having a pheromone system, which cell expresses (a)
a heterologous surrogate of a yeast pheromone system protein, said
surrogate, under at least some conditions, performing in the
pheromone system of the yeast cell a function naturally performed
by the corresponding yeast pheromone system protein, and (b) a
heterologous peptide, whereby if said peptide modulates the
interaction of said surrogate with said pheromone system, said
modulation is a selectable or screenable event.
2. The yeast cell of claim 1 wherein the endogenous pheromone
system protein is not produced in functional form.
3. The yeast cell of claim 1 wherein the peptide is secreted by the
cell into the periplasmic space, from which it interacts with said
surrogate.
4. The yeast cell of claim 3, wherein the peptide is expressed in
the form of a precursor peptide comprising a cleavable leader
peptide and a mature peptide, and the leader peptide is
substantially homologous to the leader peptide of the wild-type
pheromone of said cell.
5. The yeast cell of claim 4 wherein the wild-type leader peptide
is that of the Saccharomyces cerevisiae .alpha. factor or
a-factor.
6. The yeast cell of claim 4 in which the wild-type pheromone is
not secreted.
7. The yeast cell of claim 3 wherein the peptide is also expressed
in a nonsecretory form.
8. The yeast cell of claim 1 wherein the cell is a mutant strain
having a reduced propensity, relative to the wild-type strain, to
have its pheromone signal pathway desensitized through repeated or
prolonged stimulation thereof.
9. The yeast cell of claim 8 in which the SST2 gene is not
functionally expressed.
10. The yeast cell of claim 1, in which the FAR1 gene is not
functionally expressed.
11. The yeast cell of claim 1, further comprising a selectable
marker that is activated by the pheromone signal pathway.
12. The yeast cell of claim 11, said selectable marker comprising a
pheromone-responsive promoter which is substantially homologous
with an endogenous pheromone-responsive promoter, operably linked
to a foreign selectable gene.
13. The yeast cell of claim 12 wherein the selectable gene is an
IGP dehydratase gene.
14. The yeast cell of claim 12 wherein the homologous wild-type
promoter is the FUS1 promoter.
15. The yeast cell of claim 1 wherein the cells belong to the
species Saccharomyces cerevisiae.
16. The yeast cell of claim 1 in which the pheromone system protein
is a farnesyltransferase.
17. The yeast cell of claim 1 in which the pheromone system protein
is a carboxymethyltransferase.
18. The yeast cell of claim 1 in which the pheromone system protein
is a kinase.
19. The yeast cell of claim 1 wherein the yeast pheromone system
protein is a protease involved in the production of the mature form
of the yeast pheromone, through the cleavage of a precursor
protein, and the surrogate is also a protease.
20. The yeast cell of claim 19 wherein the precursor protein
produced in the cell is itself a surrogate of the yeast pheromone
precursor protein, and said surrogate precursor protein has an
amino acid sequence comprising a recognition site recognized by the
surrogate protease, said recognition site differing from that
recognized by the yeast pheromone system protease, but said
surrogate precursor protein is cleaved by the surrogate protease to
produce the mature form of the yeast pheromone.
21. The yeast cell of claim 20 wherein the wild type yeast
pheromone precursor protein is not produced.
22. The yeast cell of claim 1 wherein the yeast pheromone system
protein is an ABC transporter involved in the membrane transport of
the yeast pheromone, and the surrogate is also an ABC
transporter.
23. The yeast cell of claim 22 wherein the surrogate transports the
yeast pheromone unless the peptide interferes with such
transport.
24. The yeast cell of claim 22 wherein the surrogate transports the
yeast pheromone only with the aid of said peptide.
25. The yeast cell of claim 1 wherein the yeast pheromone system
protein is the yeast pheromone receptor.
26. The yeast cell of claim 25 in which the peptide is an agonist
for the surrogate receptor.
27. The yeast cell of claim 25 in which the peptide is an
antagonist for the surrogate receptor.
28. The yeast cell of claim 25 wherein the G.alpha. subunit of the
G protein is chimeric.
29. The yeast cell of claim 28 wherein the amino terminal portion
of the G.alpha. subunit is substantially homologous with the
G.alpha. subunit of a yeast G protein and the remainder is
substantially homologous with the corresponding portion of a
G.alpha. subunit of a heterologous G protein.
30. The yeast cell of claim 1 in which the pheromone system protein
is a cyclin.
31. The yeast cell of claim 30, said yeast cell further containing
a non-pheromone-responsive screenable marker.
32. A yeast culture comprising a plurality of yeast cells according
to claim 1, said yeast cells collectively expressing a peptide
library.
33. A method of assaying a peptide for modulation of the activity
of a non-yeast surrogate for a pheromone system protein which
comprises providing yeast cells according to claim 1, which cells
functionally express said surrogate and said peptide, and
determining whether the pheromone signal pathway is activated or
inhibited by said peptide.
34. The method of claim 33 in which the cells comprise a
pheromone-responsive selectable marker, and cells are selected for
expression of a peptide having the desired activating or inhibiting
effect.
35. The method of claim 33 in which the cells comprise a
pheromone-responsive screenable marker, and cells are screened for
expression of a peptide having the desired activating or inhibiting
effect.
36. A method of assaying a peptide library for activity of a
non-yeast pheromone system protein surrogate which comprises
providing a yeast culture according to claim 32, whose cells each
functionally express said surrogate and a peptide of said library,
said culture collectively expressing the entire peptide library,
and determining whether the pheromone signal pathway is activated
or inhibited by said peptides in each of the cells of said
culture.
37. The method of claim 34 in which the surrogate is human Mdr1,
the cells grow on histidine-free media only if the surrogate
transports .alpha.-factor, the cells are galactose-sensitive only
if the surrogate transports .alpha.-factor, and endogenous
pleiotropic drug resistance genes have been inactivated.
38. The yeast cell of claim 25 wherein the surrogate receptor is
the C5a receptor.
Description
[0001] This application is a continuation-in-part of Attorney
Docket No. FOWLKES=2B filed Sep. 20, 1994, which is a
continuation-in-part of Ser. No. 08/190,328, filed Jan. 31, 1994,
which is a continuation-in-part of Ser. No. 08/041,431, filed Mar.
31, 1993, all hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the screening of drugs,
especially random peptides, in yeast cells for the ability to
interact with proteins involved in the post-translational
modification, transport of and response to yeast pheromones or
substitutes therefor.
[0004] 2. Description of the Background Art
[0005] Drug Screening
[0006] The identification of biological activity in new molecules
has historically been accomplished through the use of in vitro
assays or whole animals. Intact biological entities, either cells
or whole organisms, have been used to screen for anti-bacterial,
anti-fungal, anti-parasitic and anti-viral agents in vitro.
Cultured mammalian cells have also been used in screens designed to
detect potential therapeutic compounds. A variety of bioassay
endpoints are exploited in mammalian cell screens including the
stimulation of growth or differentiation of cells, changes in cell
motility, the production of particular metabolites, the expression
of specific proteins within cells, altered protein function, and
altered conductance properties. Cytotoxic compounds used in cancer
chemotherapy have been identified through their ability to inhibit
the growth of tumor cells in vitro and in vivo. In addition to
cultures of dispersed cells, whole tissues have served in
bioassays, as in those based on the contractility of muscle.
[0007] In vitro testing is a preferred methodology in that it
permits the design of high-throughput screens: small quantities of
large numbers of compounds can be tested in a short period of time
and at low expense. Optimally, animals are reserved for the latter
stages of compound evaluation and are not used in the discovery
phase; the use of whole animals is labor-intensive and extremely
expensive.
[0008] Microorganisms, to a much greater extent than mammalian
cells and tissues, can be easily exploited for use in rapid drug
screens. Yeast provide a particularly attractive test system;
extensive analysis of this organism has revealed the conservation
of structure and function of a variety of proteins active in basic
cellular processes in both yeast and higher eukaryotes.
[0009] The search for agonists and antagonists of cellular
receptors has been an intense area of research aimed at drug
discovery due to the elegant specificity of these molecular
targets. Drug screening has been carried out using whole cells
expressing functional receptors and, recently, binding assays
employing membrane fractions or purified receptors have been
designed to screen compound libraries for competitive ligands. Duke
University, WO92/05244 (Apr. 2, 1992) describes the expression of
mammalian G protein-coupled receptors in yeast and a means of
identifying agonists and antagonists of those receptors using that
organism.
[0010] In addition, yeast are, of course, used in the discovery of
antifungal compounds; Etienne et al. (1990) describe the use of
Saccharomyces cerevisiae mutant strains, made highly sensitive to a
large range of antibiotics, for the rapid detection of
antifungals.
[0011] Yeast Pheromone System Proteins and Their Metabolic
Function
[0012] Haploid yeast cells are able not only to grow vegetatively,
but also to mate to form a diploid cell. The two mating types
("sexes") of haploid cells are designated a and .alpha.. The a
cells produce the dodecapeptide a-factor, and the .alpha. cells,
the tridecapeptide .alpha.-factor. Because a-factor and
.alpha.-factor elicit a mating response in the yeast cell of the
opposite "sex", they are called "pheromones". These pheromones, as
well as other proteins specifically involved in the production or
transport of, or response to, pheromones, are considered "pheromone
system proteins".
[0013] The gene encoding a-factor pheromone, like the
.alpha.-factor receptor gene, is an a cell-specific gene; a
cell-specific genes are only expressed in a cells. The gene
encoding .alpha.-factor pheromone, like the a-factor receptor gene,
is an .alpha. cell-specific gene; .alpha. cell-specific genes are
only expressed in .alpha. cells. Other yeast genes belong to a
haploid-specific gene set and are expressed in haploid cells (a
cells or .alpha. cells) but not in diploid (a/.alpha.) cells. In
addition, there exists a diploid cell-specific gene set, including
those genes involved in sporulation.
[0014] In eukaryotic cells, RNA polymerase II promoters contain a
specific sequence (the TATA box) to which the transcription factor
TFIID (TATA binding protein or TBP) binds. An active transcription
initiation complex includes TFIID, accessory initiation proteins,
and RNA Pol II. As in higher eukaryotic cells, the TATA box is an
essential control sequence in yeast promoters. Yeast
TATA-box-binding protein (TBP) was identified by its ability to
substitute in function for mammalian TFIID [Buratowski et al.,
Nature 334, 37 (1988); Cavallini et al., Nature 334, 77 (1988)].
With only a few apparent exceptions [transcription of some
glycolytic enzyme genes, see Struhl, Mol. Cell. Biol. 6, 3847
(1986) and Ogden et al., Mol. Cell Biol. 6, 4335 (1986)]
transcription of yeast genes requires the proximal TATA box element
and TFIID binding for initiation of transcription. Also required
for efficient transcription are gene-specific activator proteins;
the precise mechanism whereby these gene-specific regulatory
proteins influence transcription has not been completely
elucidated.
[0015] MCM1p (encoded in the MCM1 gene) is a non-cell-type-specific
transcription factor in yeast. MCM1p acts alone or in concert with
other regulatory proteins to control expression of a- and
.alpha.-cell specific genes. Yeast mating type loci encode the
regulatory proteins that contribute to the control of cell
type-specific expression. These proteins are Mata1p (encoded by the
MATa gene) and Mat.alpha.1p and Mat.alpha.2p (encoded by the
MAT.alpha. locus). MCM1p activates transcription of a-specific
genes by binding to an upstream activation sequence (UAS) located
in the control region of a-specific genes. Mat.alpha.1p and MCM1p
interact to enhance each other's binding to specific UAS binding
sites to activate .alpha.-cell-specific gene transcription in
.alpha.-cells. Mat.alpha.2p associates with MCM1p to repress
a-specific gene transcription in .alpha.-cells. In diploid
(a/.alpha.) cells, Mat.alpha.1p and Mat.alpha.2p associate to
repress the transcription of haploid-specific genes. The
Mat.alpha.1p/Mat.alpha.2p regulatory entity is found only in
diploid cells.
[0016] Yeast contain two genes encoding the .alpha.-factor
pheromone, MF.alpha.1 and MF.alpha.2. Analysis of yeast bearing
mutations in these sequences indicates that MF.alpha.1 gives rise
to the majority of .alpha.-factor produced by cells. Expression
occurs at a higher level from MF.alpha.1 than from MF.alpha.2
(Kurjan, Mol. Cell. Biol. 5, 787 (1985). The MF.alpha.1 gene of
yeast encodes a 165 aa precursor protein containing an 85 aa leader
sequence at the N-terminus. The leader includes a 19 aa signal
sequence and a 66 aa sequence which contains sites for the addition
of three oligosaccharide side chains (Kurjan and Herskowitz, Cell
39, 933 (1982); Singh et al. Nuc. Acids Res. 11, 4049 (1983);
Julius et al. Cell 36, 309 (1984). Four tandem copies of the 13 aa
.alpha.-factor are present in the C-terminal portion of the
precursor; 6-8 aa spacer peptides precede the .alpha.-factor
sequences (see FIG. 2).
[0017] After translocation of the nascent .alpha.-factor
polypeptide to the ER, the signal sequence is cleaved from the
precursor protein to yield pro-.alpha.-factor (Waters et al. J.
Biol. Chem. 263, 6209 (1988). The core N-linked carbohydrate is
added to three sites in the N-terminus of pro-.alpha.-factor (Emter
et al. Biochem. Biophys. Res. Commun. 116, 822 (1983); Julius et
al. Cell 36, 309 (1984); Julius et al. Cell 37, 1075 (1984).
Additional glycosylation occurs in the Golgi prior to cleavage of
pro-.alpha.-factor by the KEX2 endopeptidase. This enzyme cleaves
within each of the spacer repeats leaving a Lys-Arg sequence
attached to the C-terminus of .alpha.-factor peptide (Julius et al.
Cell 37, 1075 (1984). The Lys-Arg sequence is removed by the action
of the KEX-1 protease (Dmochowska et al. Cell 50, 573 (1987). The
additional spacer residues present at the N-terminus of
.alpha.-factor peptide are removed by the dipeptidyl aminopeptidase
encoded by STE13 (Julius et al. Cell 32, 839 (1983). Four
.alpha.-factor peptides are released from each precursor protein
via he proteolytic processing outlined above and the mature
.alpha.-factor is secreted from the cell.
[0018] Precursors of the 12 aa mature a-factor peptide are encoded
in the MFa1 and MFa2 genes and are 36 aa and 38 aa residues,
respectively (for schematic of MFa1 gene see FIG. 5). The
precursors contain one copy of a-factor and the products of the two
genes differ in sequence at one amino acid. The two forms of
a-factor are produced in equal amounts by a cells (Manney et al. in
Sexual interactions in eukaryotic microbes, p21, Academic Press,
New York (1981).
[0019] Processing of a-factor entails a process that differs in
every detail from that of .alpha.-factor. The processing of
a-factor begins in the cytosol and involves the farnesylation of
the C-terminal cysteine residue near the carboxyl terminus (-CVIA)
by a farnesyl transferase (Schafer et al. Science 245, 379 (1989);
Schafer et al. Science 249, 1133 (1990). The .alpha. and .beta.
subunits of the farnesyl transferase are encoded by the RAM2 and
RAM1 genes, respectively (He et al. Proc. Natl. Acad. Sci. 88,
11373 (1991). Subsequent to farnesylation is the proteolytic
removal of the three amino acids that are C-terminal to the
modified cysteine by a membrane-bound endoprotease. Next, the
carboxy-terminal farnesylated cysteine residue is modified further:
the carboxyl group is methylated by the product of the STE14 gene.
STE14p is a membrane-bound S-farnesyl-cysteine carboxyl methyl
transferase (Hrycyna et al. EMBO. J. 10, 1699 (1991). The
mechanisms of the N-terminal processing of a-factor have not been
elucidated. After processing of the precursors is complete, mature
a-factor is transported to the extracellular space by the product
of the STE6 gene (Kuchler et. al. EMBO J. 8, 3973 (1989), an
ATP-binding cassette (ABC) transporter.
[0020] In normal S. cerevisiae (budding yeast) a cells, the
.alpha.-factor binds the G protein-coupled membrane receptor STE2.
The G protein dissociates into the G.sub..alpha. and
G.sub..beta..gamma. subunits, and the G.sub..beta..gamma. binds an
unidentified effector, which in turn activates a number of genes.
STE20, a kinase, activates STE5, a protein of unknown function.
STE5 activates STE11 kinase, which stimulates STE7 kinase, which
induces the KSS1 and/or FUS3 kinases. These switch on expression of
the transcription factor STE12. STE12 stimulates expression of a
wide variety of genes involved in mating, including FUS1 (cell
fusion), FAR1 (cell-cycle arrest), STE2 (the receptor), MFA1 (the
pheromone), SST2 (recovery), KAR3 (nuclear fusion) and STE6
(pheromone secretion). Other genes activated by the pathway are
CHS1, AG.alpha.1, and KAR3. The multiply tandem sequence TGAAACA
has been recognized as a "pheromone response element" found in the
5'-flanking regions of many of the genes of this pathway.
[0021] One of the responses to mating pheromone is the transient
arrest of the yeast cell in the G1 phase of the cell cycle. This
requires that all three G1 cyclins (CLN1, CLN2, CLN3) be
inactivated. It is believed that FUS3 inactivates CLN3, and FAR1
inhibits CLN2. (The product responsible for inactivating CLN1 is
unknown).
[0022] The growth arrest is terminated by a number of different
mechanisms. First, the .alpha.-factor receptor is internalized
following binding of the pheromone, resulting in a transient
decrease in the number of pheromone binding sites. Second, the
C-terminal tail of the receptor is phosphorylated consequent to
ligand binding, resulting in uncoupling of the receptor from the
transducing G proteins. Third, pheromone-induced increases in
expression of GPA1p (the G.alpha.-subunit of the heterotrimeric G
protein) increase the level of the a subunit relative to the
G.sub..beta. and G.sub..gamma. subunits, resulting in reduction in
the level of free G.sub..beta..gamma. and consequent inactivation
of the pheromone response pathway. Additional mechanisms include
induction of the expression of SST2 and BAR1 and phosphorylation of
the .alpha. subunit (perhaps by SVG1).
[0023] Signaling is inhibited by expression of a number of genes,
including CDC36, CDC39, CDC72, CDC73, and SRM1. Inactivation of
these genes leads to activation of the signaling pathway.
[0024] A similar pheromone signaling pathway may be discerned in a
cells, but the nomenclature is different in some cases (e.g., STE3
instead of STE2).
[0025] Other yeast also have G protein-mediated mating factor
response pathways. For example, in the fission yeast S. pombe, the
M factor binds the MAP3 receptor, or the P-factor the MAM2
receptor. The dissociation of the G protein activates a kinase
cascade (BYR2, BYR1, SPK1), which in turn stimulates a
transcription factor (STE11). However, in S. pombe, the G.alpha.
subunit transmits the signal, and there are of course other
differences in detail.
[0026] Pheromone Pathway Mutants
[0027] The effects of spontaneous and induced mutations in
pheromone pathway genes have been studied. These include the
.alpha.-factor (MF.alpha.1 and MF.alpha.2) genes, see Kurjan, Mol.
Cell. Biol., 5:787 (1985); the a-factor (MFa1 and MFa2) genes, see
Michaelis and Herskowitz, Mol. Cell. Biol 8:1309 (1988); the
pheromone receptor (STE2 and STE3) genes, see Mackay and Manney,
Genetics, 76:273 (1974), Hartwell, J. Cell. iol., 85:811 (1980),
Hagen, et al., P.N.A.S. (USA), 83:1418 (1986); the FAR1 gene, see
Chang and Herskowitz, Cell, 63:999 (1990); and the SST2 gene, see
Chan and Otte, Mol. Cell. Biol., 2:11 (1982).
[0028] Expression of Foreign Proteins in Yeast Cells
[0029] A wide variety of foreign proteins have been produced in S.
cerevisiae, either solely in the yeast cytoplasm or through
exploitation of the yeast secretory pathway (Kingsman et al.
TIBTECH 5, 53 (1987). These proteins include, as examples,
insulin-like growth factor receptor (Steube et al. Eur. J. Biochem.
198, 651 (1991), influenza virus hemagglutinin (Jabbar et al. Proc.
Natl. Acad. Sci. 82, 2019 (1985), rat liver cytochrome P 450 (Oeda
et al. DNA 4, 203 (1985) and functional mammalian antibodies (Wood
et al. Nature 314, 446 (1985). Use of the yeast secretory pathway
is preferred since it increases the likelihood of achieving
faithful folding, glycosylation and stability of the foreign
protein. Thus, expression of heterologous proteins in yeast often
involves fusion of the signal sequences encoded in the genes of
yeast secretory proteins (e.g., .alpha.-factor pheromone or the
SUC2 [invertase] gene) to the coding region of foreign protein
genes.
[0030] A number of yeast expression vectors have been designed to
permit the constitutive or regulated expression of foreign
proteins. Constitutive promoters are derived from highly expressed
genes such as those encoding metabolic enzymes like
phosphoglycerate kinase (PGK1) or alcohol dehydrogenase I (ADH1)
and regulatable promoters have been derived from a number of genes
including the galactokinase (GAL1) gene. In addition,
supersecreting yeast mutants can be derived; these strains secrete
mammalian proteins more efficiently and are used as "production"
strains to generate large quantities of biologically active
mammalian proteins in yeast (Moir and Davidow, Meth. in Enzymol.
194, 491 (1991).
[0031] A variety of heterologous proteins have been expressed in
yeast cells as a means of generating the quantity of protein
required for commercial use or for biochemical study (Kingsman et
al. TIBTECH 5, 53 (1987). In addition, a number of mammalian
proteins have been expressed in yeast in order to determine whether
the proteins (1) will functionally substitute for cognate proteins
normally expressed within that organism or (2) will interact with
accessory yeast proteins to accomplish a specific function. Thus it
has been determined that a human TBP with altered binding
specificity will function to initiate transcription in yeast
[Strubin an Struhl, Cell 68, 721 (1992)]. In addition, mammalian
steroid hormone receptors [Metzger et al. (1988); Schena and
Yamamoto (1988)] and human p53 [Schrer and Iggo, Nuc. Acids Res.
20, 1539 (1992)] were shown to activate transcription in yeast.
[0032] Expression in yeast of the gag-pol gene of HIV-1 results in
the processing of the gag protein precursor to yield the products
which normally arise within the virion; processing in yeast, as in
the virus, is due to the action of the protease encoded within the
gag-pol gene (Kramer et al. Science 231,1580 (1986).
[0033] A number of mammalian ABC transporters have been expressed
in yeast to determine their ability to substitute for yeast Ste6p
in the transport of pheromone. The mammalian proteins thus far
tested include human Mdr1 (Kuchler and Thorner, Proc. Natl. Acad.
Sci. 89, 2302 (1992)) and murine Mdr3 (Raymond et al. Science 256,
232 (1992), proteins involved in multidrug resistance; in addition,
a chimeric protein containing human CFTR (cystic fibrosis
transmembrane conductance regulator) and yeast STE6 sequence has
been shown to transport a-factor pheromone in yeast (Teem et al.
Cell 73, 335 (1993)
[0034] An a cell may be engineered to produce the a-factor
receptor, and an .alpha. cell to make .alpha.-factor receptor.
Nakayama, et al., EMBO J., 6:249-54 (1987); Bender and Sprague,
Jr., Genetics 121: 463-76 (1989).
[0035] Heterologous G protein-coupled receptors have been
functionally expressed in S. cerevisiae. Marsh and Hershkowitz,
Cold Spring Harbor Symp., Quant. Biol., 53: 557-65 (1938) replaced
the S. cerevisiae STE2 with its homologue from S. Kluyven. More
dramatically, a mammalian beta-adrenergic receptor and G.alpha.
subunit have been expressed in yeast and found to control the yeast
mating signal pathway. King, et al., Science, 250: 121-123
(1990).
[0036] Duke University, WO92/05244 (Apr. 2, 1992) describes a
transformed yeast cell which is incapable of producing a yeast G
protein .alpha. subunit, but which has been engineered to produce
both a mammalian G protein .alpha. subunit and a mammalian receptor
which is "coupled to" (i.e., interacts with) the aforementioned
mammalian G protein .alpha. subunit. Specifically, Duke reports
expression of the human beta-2 adrenergic receptor (h.beta.AR), a
seven transmembrane receptor (STR), in yeast, under control of the
GAL1 promoter, with the h.beta.AR gene modified by replacing the
first 63 base pairs of coding sequence with 11 base pairs of
noncoding and 42 base pairs of coding sequence from the STE2 gene.
(STE2 encodes the yeast .alpha.-factor receptor). Duke found that
the modified h.beta.AR was functionally integrated into the
membrane, as shown by studies of the ability of isolated membranes
to interact properly with various known agonists and antagonists of
h.beta.AR. The ligand binding affinity for yeast-expressed
h.beta.AR was said to be nearly identical to that observed for
naturally produced h.beta.AR.
[0037] Duke co-expressed a rat G protein .alpha. subunit in the
same cells, yeast strain 8C, which lacks the cognate yeast protein.
Ligand binding resulted in G protein-mediated signal
transduction.
[0038] Duke teaches that these cells may be used in screening
compounds for the ability to affect the rate of dissociation of
G.alpha. from G.beta..gamma. in a cell. For this purpose, the cell
further contains a pheromone-responsive promoter (e.g. BAR1 or
FUS1), linked to an indicator gene (e.g. HIS3 or LacZ). The cells
are placed in multi-titer plates, and different compounds are
placed in each well. The colonies are then scored for expression of
the indicator gene.
[0039] Duke's yeast cells do not, however, actually produce the
compounds to be screened. As a result, only a relatively small
number of compounds can be screened, since the scientist must
ensure that a given group of cells is contacted with only a single,
known compound.
[0040] Yeast have been engineered to express foreign polypeptide
variants to be tested as potential antagonists of mammalian
receptors. Libraries encoding mutant glucagon molecules were
generated through random misincorporation of nucleotides during
synthesis of oligonucleotides containing the coding sequence of
mammalian glucagon. These libraries were expressed in yeast and
culture broths from transformed cells were used in testing for
antagonist activity on glucagon receptors present in rat hepatocyte
membranes (Smith et al. 1993).
[0041] Drugs which overcome the multiple drug resistance (MDR) of
cancer cells may be identified by using transformed yeast cells
expressing P-glycoprotein (Suntory Ltd., patent application JP
2257873 entitled "Multiple drug resistance-relating gene-comprises
P-glycoprotein accumulated in cell membrane part of transformed
yeast"). The drugs were not produced by the yeast cells in
question.
[0042] A yeast strain has been derived to allow the identification
of inhibitors of protein farnesyltransferase which exhibit activity
against mammalian Ras and which may therefore function as antitumor
drugs (Hara et al. 1993).
[0043] Genetic Markers in Yeast Strains
[0044] Yeast strains that are auxotrophic for histidine (HIS3) are
known, see Struhl and Hill), Mol. Cell. Biol., 7:104 (1987);
Fasullo and Davis, Mol. Cell. Biol., 8:4370 (1988). The HIS3
(imidazoleglycerol phosphate dehydratase) gene has been used as a
selective marker in yeast. See Sikorski and Heiter, Genetics,
122:19 (1989); Struhl et al., P.N.A.S. 76:1035 (1979); and, for
FUS1-HIS3 fusions, see Stevenson, et al., Genes Dev., 6:1293
(1992). Peptide Libraries. Peptide libraries are systems which
simultaneously display, in a form which permits interaction with a
target, a highly diverse and numerous collection of peptides. These
peptides may be presented in solution (Houghten), or on beads
(Lam), chips (Fodor), bacteria (Ladner U.S. Pat. No. 5,223,409),
spores (Ladner U.S. Pat. No. '409), plasmids (Cull) or on phage
(Scott, Devlin, Cwirla, Felici, Ladner '409). Many of these systems
are limited in terms of the maximum length of the peptide or the
composition of the peptide (e.g., Cys excluded). Steric factors,
such as the proximity of a support, may interfere with binding.
Usually, the screening is for binding in vitro to an artificially
presented target, not for activation or inhibition of a cellular
signal transduction pathway in a living cell. While a cell surface
receptor may be used as a target, the screening will not reveal
whether the binding of the peptide caused an allosteric change in
the conformation of the receptor.
[0045] Ladner, U.S. Pat. No. 5,096,815 describes a method of
identifying novel proteins or polypeptides with a desired DNA
binding activity. Semi-random ("variegated") DNA encoding a large
number of different potential binding proteins is introduced, in
expressible form, into suitable host cells. The target DNA sequence
is incorporated into a genetically engineered operon such that the
binding of the protein or polypeptide will prevent expression of a
gene product that is deleterious to the gene under selective
conditions. Cells which survive the selective conditions are thus
cells which express a protein which binds the target DNA. While it
is taught that yeast cells may be used for testing, bacterial cells
are preferred. The interactions between the protein and the target
DNA occur only in the cell (and then only in the nucleus), not in
the periplasm or cytoplasm, and the target is a nucleic acid, and
not a pheromone system protein surrogate.
[0046] Substitution of random peptide sequences for functional
domains in cellular proteins permits some determination of the
specific sequence requirements for the accomplishment of function.
Though the details of the recognition phenomena which operate in
the localization of proteins within cells remain largely unknown,
the constraints on sequence variation of mitochondrial targeting
sequences and protein secretion signal sequences have been
elucidated using random peptides (Lemire et al., J. Biol. Chem.
264, 20206 (1989) and Kaiser et al. Science 235, 312 (1987),
respectively).
[0047] All references cited in this specification are hereby
incorporated by reference. No admission is made that any reference
constitutes prior art.
SUMMARY OF THE INVENTION
[0048] In the present invention, a yeast cell is engineered to
express an exogenous protein which is, however, capable of
substituting for a yeast protein which is involved in the
post-translational modification, transport, recognition or signal
transduction of a yeast pheromone, sufficiently, to be able, at
least under some circumstances, to carry out that role of the yeast
protein. For the sake of convenience, these yeast proteins will be
referred to as "pheromone system proteins" (PSP), and their cognate
non-yeast proteins as PSP surrogates.
[0049] The pheromone system of a yeast cell is thus subverted so
that the response of the cell to a yeast pheromone is at least
partially determined by the activity of the surrogate PSP. In a
preferred embodiment, the cognate yeast PSP is not produced in
functional form, so that the response is essentially entirely
dependent on the activity of the surrogate PSP.
[0050] Such yeast cells may be used to identify drugs which inhibit
or activate, to a detectable degree, the ability of the surrogate
to substitute for the cognate yeast PSP. To screen for an
inhibitor, a normally functional surrogate is expressed, and the
presence of an inhibitor is indicated by a depression of the
cellular response. To screen for an activator, a surrogate
functional in yeast, or one normally not functional in yeast but
which is activatable (the latter is preferred, to minimize
background) is used, and the activator is detected through its
elevation of the cellular response.
[0051] In a preferred embodiment, the candidate drug is a peptide,
and the yeast cell is engineered to express the candidate drug as
well as the surrogate PSP.
[0052] Another consideration is that with wild-type yeast cells, to
achieve pheromone secretion and response, both .alpha. and a cells
must be provided. In some preferred embodiments, .alpha. cells are
engineered to express .alpha.-factor receptor or a cells are
engineered to express a-factor receptor. These modified cells may
be considered "autocrine" because they are "self-stimulatory".
Cells which express both a surrogate for the pheromone receptor,
and a heterologous peptide agonist for the surrogate receptor, are
also considered "autocrine", because they will respond to the
co-produced agonist.
[0053] The classes of PSPs are numerous. However, some examples may
be helpful. Farnesyltransferases and carboxymethyltransferases.
After expression, a-factor is farnesylated by RAM1p and RAM2p and
carboxymethylated by Ste14p. These modifications are required for
activity.
[0054] RAM1p and RAM2p are homologous to the subunits of the
heterodimeric mammalian farnesyltransferase, which itself is
necessary for membrane association of mammalian Ras proteins. If a
yeast cell is engineered to express the mammalian
farnesyltransferase, it may be used to identify drugs which
interact with that enzyme by determining whether a functional
a-factor is produced. Similarly, Ste14p is homologous to mammalian
carboxymethyltransferases, which play regulatory roles in
controlling the function of low molecular weight G proteins (Ras,
Rho, Rab).
[0055] Proteases. The PSP may be a yeast protease, such as KEX2,
STE13 or KEX1. Yeast .alpha.-factor pheromone is generated through
the controlled and limited proteolysis of precursor proteins by
these proteases. A yeast cell may be engineered to express an
inactive precursor of yeast .alpha.-factor in which a peptide
linker, corresponding to the cleavage site of a surrogate non-yeast
protease, is incorporated so that cleavage will liberate mature
.alpha.-factor (or its functional homologue). For example, the PSP
surrogate may be HIV protease, with the cleavage site of HIV
protease being substituted for the yeast protease cleavage sites in
the .alpha.-factor precursor. The Precursor and the HIV protease
are co-expressed in the yeast cell. Proteolysis by HIV protease
will give rise to production of mature .alpha.-factor and
initiation of signal transduction. This system may be used to
identify inhibitors of HIV protease.
[0056] Preferably, unlike yeast cells occurring in nature, the
yeast cell is engineered not only to express the .alpha.-factor
precursor, but also the .alpha.-factor receptor, so that a single
haploid type of yeast is all that is required to conduct the
assay.
[0057] ABC Transporters. Ste6 is the yeast ABC transporter
necessary for the export of a-factor. The yeast cell is engineered
to express both a-factor and a foreign ABC transporter. This
transporter may be one which is not, by itself, able to transport
a-factor, but which in the presence of a drug of interest, is
capable of doing so, or it may be one which is already
functional.
[0058] Preferably, the yeast cell is engineered to express not only
a-factor, but also the a-factor receptor.
[0059] G Protein-Coupled Receptors. The PSP may be a yeast
pheromone receptor. The surrogate is a non-yeast, G protein-coupled
receptor. In order to achieve coupling to the pheromone signal
transduction pathway, it may be necessary to provide a foreign or
chimeric G.sub..alpha. or G.sub..beta..gamma. subunit.
[0060] The engineered yeast cell may be used to screen for agonists
as well as antagonists. When used to screen for agonists, it is
preferable that the yeast pheromone not be produced in functional
form.
[0061] Protein Kinases. The PSP may be a protein kinase, such as
the FUS1, KSS1, STE11 or STE7 proteins, which participate in the
cellular response to pheromones. The PSP surrogate would be, e.g.,
a mammalian, mitogen-activated protein kinase. Yeast cells
engineered to express the surrogate protein kinase could be used to
screen for activators or inhibitors thereof.
[0062] Cyclins. The PSP may be a cyclin, such as CLN1, CLN2 or
CLN3. The cyclins regulate the progression of cells through the
cell cycle. The human C, D1 and E cyclins are capable of
substituting functionally for the CLN proteins of yeast. Inhibitors
of mammalian cyclins may be useful in cancer chemotherapy. Yeast
cells engineered to express a surrogate cyclin may be used to
identify molecules which inhibit (or enhance) its activity.
[0063] Peptide Libraries
[0064] While others have engineered yeast cells to facilitate
screening of exogenous drugs as receptor agonists and antagonists,
the cells did not themselves produce both the drugs and the
receptors. Yeast cells engineered to produce the receptor, but that
do not produce the drugs themselves, are inefficient. To utilize
them one must bring a sufficient concentration of each drug into
contact with a number of cells in order to detect whether or not
the drug has an action. Therefore, a microtiter plate well or test
tube must be used for each drug. The drug must be synthesized in
advance and be sufficiently pure to judge its action on the yeast
cells. When the yeast cell produces the drug, the effective
concentration is higher.
[0065] In a preferred embodiment, the yeast cells collectively
produce a "peptide library", preferably including at least 10.sup.7
different peptides, so that diverse peptides may be simultaneously
assayed for the ability to interact with the PSP surrogate.
[0066] In an especially preferred embodiment, at least some
peptides of the peptide library are secreted into the periplasm,
where they may interact with the "extracellular" binding site(s) of
an exogenous receptor. They thus mimic more closely the clinical
interaction of drugs with cellular receptors. This embodiment
optionally may be further improved (in assays not requiring
pheromone secretion) by preventing pheromone secretion, and thereby
avoiding competition between the peptide and the pheromone for
signal peptidase and other components of the secretion system.
[0067] Detection of Signal Transduction
[0068] Yeast cells may also be engineered so that their pheromone
signal transduction pathways provide a more readily detectable
evidence of the activity of a suspected drug. In these embodiments,
the drug need not be a peptide produced by the same yeast cell, or
even a peptide at all.
[0069] As previously mentioned, one of the consequences of
activation of the pheromone signal pathway in wild-type yeast is
growth arrest. If one is testing for an antagonist of a G
protein-coupled receptor, or of other pheromone system proteins,
this normal response of growth arrest can be used to select cells
in which the pheromone response pathway is inhibited. That is,
cells exposed to both a known agonist and a peptide of unknown
activity will be growth arrested if the peptide is neutral or an
agonist, but will grow normally if the peptide is an antagonist.
Thus, the growth arrest response can be used to advantage to
discover peptides that function as antagonists.
[0070] However, when searching for peptides which can function as
agonists of G protein-coupled receptors, or other pheromone system
proteins, the growth arrest consequent to activation of the
pheromone response pathway is an undesirable effect for this
reason: cells that bind peptide agonists stop growing while
surrounding cells that fail to bind peptides will continue to grow
The cells of interest, then, will be overgrown or their detection
obscured by the background cells, confounding identification of the
cells of interest. To overcome this problem the present invention
teaches engineering the cell such that: 1) growth arrest does not
occur as a result of pheromone signal pathway activation (e.g., by
inactivating the FAR1 gene); and/or 2) a selective growth advantage
is conferred by activating the pathway (e.g., by transforming an
auxotrophic mutant with a HIS3 gene under the control of a
pheromone-responsive promoter, and applying selective
conditions).
[0071] It is, of course, desirable that the exogenous receptor (or
other PSP surrogate) be exposed on a continuing basis to the
peptides. Unfortunately, this is likely to result in
desensitization of the pheromone pathway to the stimulus.
Desensitization may be avoided by mutating (which may include
deleting) the SST2 gene so that it no longer produces a functional
protein, or by mutating other genes which may contribute to
desensitization, e.g., BAR1 in the case of a cells and SVG1 for
either a or .alpha. cells.
[0072] If the endogenous pheromone receptor (or other cognate PSP)
is produced by the yeast cell, the assay will not be able to
distinguish between peptides which interact with the pheromone
receptor (or other cognate PSP) and those which interact with the
exogenous receptor (or other PSP surrogate). It is therefore
desirable that the endogenous gene be deleted or otherwise rendered
nonfunctional.
[0073] The claims are hereby incorporated by reference as a further
description of the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1. Outline of successive stages in the development of
yeast autocrine systems. An outline of the normal synthesis and
release of mating pheromones is diagrammed in the upper left. Two
genes, MF.alpha.1 and MF.alpha.2, encode precursor proteins
(MF.alpha.1p and MF.alpha.2p) containing four and two repeats,
respectively, of the tridecapeptide representing mature
.alpha.-factor. These precursors are processed proteolytically in a
series of enzymatic reactions that begin with cleavage of the
signal sequence in the endoplasmic reticulum and involve both
glycosylation of the leader peptide and cleavage by the proteases
KEX2p, STE13p, and KEX1P. The result is the secretion of mature
.alpha.-factor which, upon binding to STE2p normally expressed on
the surface of a cells, elicits a number of changes in the a cells,
including growth arrest. The a cells, in turn, express two genes,
MFa1 and MFa2, which encode precursors (MFa1p and MFa2p) for
a-factor. These precursors undergo farnesylation by RAM1 and RAM2,
proteolytic trimming of the C-terminal three amino acids (by a
protein tentatively identified as RAM3p), carboxymethylation of the
newly exposed C-terminal cysteine by STE14p, and endoproteolytic
removal of the N-terminal leader sequence by an activity
provisionally identified as STE19p. Upon export of the mature
a-factor from the cell via STE6p, it binds to STE3p expressed on
the surface of .alpha. cells and stops their growth.
[0075] Stage 1 involves the development of yeast strains in which
SST2, FAR1, and HIS3 are inactivated and a suitable reporter
construct like fus1::HIS3 is integrated into the genomes of both
.alpha. and a cells. .alpha. cells are further altered by
replacement of the normally expressed STE3p with STE2p, while a
cells are further modified by replacement of the normally expressed
STE2p with STE3p. The resulting strains should show growth on
histidine-deficient media in the absence of exogenous
pheromone.
[0076] Stage 2 involves, first, inactivation of MF.alpha.1 and
MF.alpha.2 in cells and inactivation of MFa1 and MFa2 in a cells
developed in Stage 1. These modifications will result in strains
which are auxotrophic for histidine. Next, the appropriate
expression plasmid will be introduced: the expression plasmid
pADC-MF (see FIG. 4) containing an oligonucleoide encoding
.alpha.-factor should confer upon .alpha. cells the ability to grow
on histidine-deficient media; the expression plasmid pADC-MFa (see
FIG. 6) containing an oligonucleotide encoding a-factor should
enable a cells to grow on histidine-deficient media.
[0077] Stage 3 uses the cells developed in Stage 2 for the
insertion of expression plasmids. However, instead of using
plasmids which contain oligonucleotides that encode genuine
pheromone, the yeast will be transformed with expression plasmids
that contain random or semi-random oligonucleotides. Transformants
which can grow on histidine-deficient media will be expanded and
their plasmids isolated for sequencing of the inserted
oligonucleotide.
[0078] FIG. 2. Diagram of the plasmid used for mutagenesis of
MF.alpha.1. A 1.8 kb EcoRI fragment containing MF.alpha.1 is cloned
into the EcoRI site of pALTER such that single-stranded DNA
containing the MF.alpha.1 minus strand can be synthesized. The
diagram illustrates the different regions of MF.alpha.1, including
the promoter, transcription terminator, and different domains of
the precursor protein: the signal peptide, the pro peptide, the
four repeats of mature .alpha.-factor, and the three spacers which
separate these repeats. Above the block diagram of the regions of
MF.alpha.1 are the amino acid sequences (SEQ ID NO:1) of the signal
peptide and the pro peptide; below it are those of the pheromone
repeats and the spacers (SEQ ID NO:2). The sites of proteolytic
processing of the precursor protein are indicated by arrows, with
each proteolytic activity represented by a different arrow, as
indicated in the figure.
[0079] FIG. 3. Diagram of the plasmids used in the construction of
the MF.alpha. expression cassette. pAAH5 contains the ADC1 promoter
which will be used to drive expression of synthetic
oligonucleotides inserted into the MF.alpha. expression cassette.
The 1.5 kb BamHI to HindIII fragment containing the ADC1 promoter
will be cloned into pRS426, a plasmid which functions as a
high-copy episome in yeast, to yield pRS-ADC. pRS-ADC will be the
recipient of MF.alpha.1 sequences which have been mutated as
follows: the region of MF.alpha.1 which encodes mature
.alpha.-factor will be replaced with restriction sites that can
accept oligonucleotides with Af1 II and BglII ends. Insertion of
oligonucleotides with AflII and BglII ends will yield a plasmid
which encodes a protein containing the MF.alpha.1 signal and leader
sequences upstream of the sequence encoded by the oligonucleotide.
The MF.alpha.1 signal and leader sequences should direct the
processing of this precursor protein through the pathway normally
used for the secretion of mature .alpha.-factor.
[0080] FIG. 4. Diagram of constructs used for the expression of
random oligonucleotides in the context of MF.alpha.1.
Oligonucleotides containing a region of 39 random base pairs (shown
at the top of the figure) will be cloned into the AflII and BglII
sites of the MF.alpha.1 expression cassette. These oligonucleotides
will encode the six amino acids immediately N-terminal to the first
repeat of the .alpha.-factor in MF.alpha.1, followed in succession
by a tridecapeptide of random sequence and a stop codon. Yeast
transformed with these constructs and selected for an ability to
grow on media deficient in uracil will use the ADC1 promoter to
express a protein consisting of the MF.alpha.1 leader (both pre and
pro peptides) followed by 13 random amino acids. Processing of the
leader sequences will result in secretion of the tridecapeptide. A
nucleotide sequence (SEQ ID NO:3) is presented upstream of the
leader sequence and a second nucleotide sequence (SEQ ID NO:4)
containing AflII, XhoI and BglII sites and coding for a peptide
with an amino acid sequence (SEQ ID NO:5) is presented downstream
from the leader sequence. A nucleotide sequence (SEQ ID NO:6)
containing an AflII and a BclI site encodes for a peptide with an
amino acid sequence (SEQ ID NO:7).
[0081] FIG. 5. Diagram of the plasmid used for mutagenesis of MFa1.
A 1.6 kb BamHI fragment containing MFa1 is cloned into the BamHI
site of pALTER such that single-stranded DNA containing the MFa1
minus strand can be synthesized. The diagram illustrates the
different regions of MFa1, including the promoter, transcription
terminator, and different domains of the precursor protein: the
leader peptide; the dodecapeptide that represents the peptide
component of mature a-factor and whose C-terminal cysteine becomes
farnesylated and carboxymethylated during processing; and the
C-terminal three amino acids that are removed during processing of
the precursor. Above the block diagram of the regions of MFa1 is
the amino acid sequence (SEQ ID NO:8) of the primary translation
product.
[0082] FIG. 6. Diagram of constructs used for the expression of
random oligonucleotides in the context of MFa1. Oligonucleotides
containing a region of 33 random base pairs (shown at the top of
the figure) will be cloned into the XhoI and AflII sites of the
MFa1 expression cassette. These oligonucleotides will encode the
seven amino acids immediately N-terminal to the first amino acid of
mature a-factor, followed in succession by a monodecapeptide of
random sequence, a cysteine which is farnesylated and
carboxymethylated during processing of the precursor, three amino
acids (VIA) which are proteolytically removed during processing,
and a stop codon. Yeast transformed with these constructs and
selected for an ability to grow on media deficient in uracil will
use the ADC1 promoter to express a precursor protein consisting of
the MFa1 leader followed by 11 random amino acids and a C-terminal
tetrapeptide CVIA. Processing of this precursor will result in
secretion of a C-terminally farnesylated, carboxymethylated
dodecapeptide which consists of 11 random amino acids and a
C-terminal cysteine. A nucleotide sequence (SEQ ID NO:9) is
presented upstream of the MFaI leader and a second nucleotide
sequence (SEQ ID NO:10) containing an AflII and a XhoI site and
encoding for a peptide with an amino acid sequence (SEQ ID NO:11)
is presented downstream from the MFaI leader. A nucleotide sequence
(SEQ ID NO:12) containing corresponding AflII and XhoI sites for
cloning into SEQ ID NO:10 encodes for a peptide with an amino acid
sequence (SEQ ID NO:13).
[0083] FIG. 7. Autocrine Mata strain secretes and responds to
signalling by .alpha.-factor.
[0084] A synthetic oligonucleotide encoding the yeast
.alpha.-factor pheromone was expressed in Mata cells. These cells
normally express the a-factor pheromone but were prevented from
doing so by deletion of the endogenous a factor-encoding genes.
Expression and release of .alpha.-factor by these cells renders
them "autocrine" with regard to pheromone signalling. The peptide
containing mature .alpha.-factor was processed within these cells
for transport through the yeast secretory pathway to the
extracellular environment. Pheromone signalling was initiated by
the binding of .alpha.-factor to the Ste2 receptor expressed in
Mata cells. Signalling by pheromone in the strain background used
in these experiments results in growth of responsive cells on media
that is deficient in histidine. Background growth of control cells
that are not expressing .alpha.-factor is prevented by increasing
concentrations of the HIS3 inhibitor, aminotriazole.
[0085] FIG. 8. Autocrine MATa strain secretes and responds to
signalling by a-factor.
[0086] Yeast a-factor was expressed from a plasmid containing a
synthetic pheromone-encoding oligonucleotide in Mata cells. The
yeast used in these experiments were made "autocrine" by
replacement of the normally expressed Ste2 protein with the
receptor for a-factor, Ste3. The a-factor peptide was processed in
these cells and transported to the extracellular environment by the
endogenous Ste6 protein, an ATP-dependent transmembrane
transporter. Pheromone signalling initiated by the a-factor
released by these cells when bound to Ste3 is indicated by the
growth of the cells on histidine-deficient media. Background growth
of control cells, which are incapable of expressing a-factor (these
a cells lack the plasmid which encodes the pheromone) is prevented
by increasing concentrations of the HIS3 inhibitor,
aminotriazole.
[0087] FIG. 9. Plasmid pYMA177 containing mutant human MDR1 (G185V
mutation).
[0088] The plasmid pYMA177 was constructed by Karl Kuchler and
permits the simultaneous overexpression of both a mutant human Mdr1
protein and the yeast a-factor pheromone precursor (Kuchler &
Thorner, Proc. Natl. Acad. Sci. 89, 2302 (1992) Cadus 1270,
containing a galactose inducible, amino-terminal myc-tagged form of
the C5a receptor, was used to transform a protease deficient strain
of yeast.
[0089] FIG. 10. Activity of a fus1 promoter in response to
signalling by human C5a expressed in autocrine strains of yeast. To
verify and quantify pheromone pathway activation upon stimulation
of the C5a receptor by C5a in yeast, the activity of the fus1
promoter was determined colorometrically using a fus1-lacZ fusion.
CY878 (MAT.alpha. tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156
gpa1 (41)-G.alpha.i2) was transformed with CADUS 1584
(pRS424-fus1-lacZ) in addition to receptor and ligand plasmids
listed below. Transformants were grown overnight in synthetic
medium lacking leucine, uracil, and tryptophan, pH 6.8, 50 mM PIPES
to an OD.sub.600 of less than 0.8 and .beta.-galactosidase activity
(Guarente 1983) was assayed.
1 Cadus 1289 + Cadus 1215 = Receptor.sup.- Ligand.sup.- = (R-L-)
Cadus 1303 + Cadus 1215 = Receptor.sup.+ Ligand.sup.- = (R+L-)
Cadus 1289 + Cadus 1297 = Receptor.sup.- Ligand.sup.+ = (R-L+)
Cadus 1303 + Cadus 1297 = Receptor.sup.+ Ligand.sup.+ = (R+L+)
Receptor refers to the human C5a receptor. Ligand refers to human
C5a.
[0090] FIG. 11. Thin Figure schematically describes three hybrids
of GPA1 and G.alpha.S. The -LLLLGAGES- sequence demarcated in GPA1
directly follows the non-conserved N-terminal domain of the
protein. The longer sequence demarcated in GPA1 encodes the "switch
region" believed to be involved in the conformational change that
occurs with nucleotide exchange upon receptor activation.
41-G.alpha.s is comprised of the N-terminal 41 amino acids of GPA1
linked to G.alpha.s sequence from which the native N-terminal
sequence has been deleted. SGS denotes a molecule comprised of the
switch region residues of GPA1 replacing those of G.alpha.s.
GPA.sub.41-SGS includes both the N-terminal and switch region
sequences of GPA1 inserted into G.alpha.s. (See Table 6 for the
exact sequence junctions used to construct these hybrid
proteins).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0091] The present invention contemplates the assaying of peptides,
especially when presented in peptide libraries, expressed in
genetically engineered yeast cells, for the ability of the peptides
to interact with pheromone system proteins and PSP surrogates
produced by those yeast cells.
[0092] For the purpose of the present invention, an "exogenous"
protein is one which sufficiently differs in amino acid sequence
from the proteins naturally produced by the yeast cell in question
so that its closest cognate is a protein produced by a cell other
than a yeast cell. The cell producing this cognate protein may be a
microbial cell (other than a yeast cell), a plant cell, or an
animal cell. If an animal cell, it may be of invertebrate (e.g.,
insect or nematode) or of vertebrate (e.g., avian, piscine or
mammalian, especially human) origin. A protein is considered to be
of, e.g., human origin, regardless of whether it is encoded by the
chromosome of a normal human, or by the genome of a virus which
infects and replicates in human cells.
[0093] An "activator" of a pheromone system protein surrogate is a
substance which, in a suitable yeast cell, causes the pheromone
system protein surrogate to become more active, and thereby
elevates the signal transduced by the native or modified pheromone
signal pathway of said cell to a detectable degree. The surrogate
may be initially nonfunctional, but rendered functional as a result
of the action of the activator, or it may be functional, and the
effect of the activator is to heighten the activity of the
surrogate. The mode of action of the activator may be direct, e.g.,
through binding the surrogate, or indirect, e.g., through binding
another molecule which otherwise interacts with the surrogate. When
the PSP surrogate is a substitute for a pheromone receptor, and the
activator takes the place of the pheromone, it is customary to
refer to the activator as an agonist of the receptor.
[0094] Conversely, an "inhibitor" of a pheromone system protein
surrogate is a substance which, in a suitable yeast cell, causes
the PSP surrogate to become less active, and thereby reduces the
transduced signal to a detectable degree. The reduction may be
complete or partial. When the PSP surrogate is a substitute for a
pheromone receptor, and the inhibitor competes with the pheromone
for binding to the receptor, it is customary to refer to the
inhibitor as an "antagonist".
[0095] The term "modulator" includes both "activators" and
"inhibitors".
[0096] A surrogate PSP protein is "functionally homologous" to a
yeast protein if, either alone or after being modified by a drug,
it is able to perform the function of the yeast PSP, or an
analogous function, within the engineered yeast cell. It is not
necessary that it be as efficient as the yeast protein, however, it
is desirable that it have at least 10% of at least one of the
pheromone system-related activities of the yeast protein. Nor is it
necessary that it have the same spectrum of action as the yeast
protein, e.g., if it is a receptor, it may respond to entirely
different ligands than does the endogenous receptor, or to some
common ligands and some new ones. The receptors of Table 2 are
considered functionally homologous with the yeast pheromone
receptors, even though they do not respond to yeast pheromones, and
may not couple to the unmodified endogenous G proteins, although
they are G protein-coupled receptors. This is considered an
"analogous function".
[0097] The PSP surrogate may be a protein which must be modified in
some way by a drug to be functional. For example, the drug could
cause an allosteric change in the PSP surrogate's conformation, or
it could cleave off a portion of the surrogate, the balance of the
protein then being a functional molecule.
[0098] The PSP surrogate may also be one which is functional only
if other modifications are made in the yeast cell, e.g., expression
of a chimeric G a subunit to interact with an exogenous G
protein-coupled receptor.
[0099] The term "substantially homologous", when used in connection
with amino acid sequences, refers to sequences which are
substantially identical to or similar in sequence, giving rise to a
homology in conformation and thus to similar biological activity.
The term is not intended to imply a common evolution of the
sequences.
[0100] Typically, "substantially homologous" sequences are at least
50%, more preferably at least 80%, identical in sequence, at least
over any regions known to be involved in the desired activity. Most
preferably, no more than five residues, other than at the termini,
are different. Preferably, the divergence in sequence, at least in
the aforementioned regions, is in the form of "conservative
modifications".
[0101] "Conservative modifications" are defined as
[0102] (a) conservative substitutions of amino acids as hereafter
defined; and
[0103] (b) single or multiple insertions or deletions of amino
acids at the termini, at interdomain boundaries, in loops or in
other segments of relatively high mobility.
[0104] Preferably, except at the termini, no more than about five
amino acids are inserted or deleted at a particular locus, and the
modifications are outside regions known to contain binding sites
important to activity.
[0105] Conservative substitutions are herein defined as exchanges
within one of the following five groups:
[0106] I. Small aliphatic, nonpolar or slightly polar residues:
Ala, Ser, Thr (Pro, Gly)
[0107] II. Polar, negatively charged residues: and their amides
Asp, Asn, Glu, Gln
[0108] III. Polar, positively charged residues: His, Arg, Lys
[0109] IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val
(Cys)
[0110] V. Large, aromatic residues: Phe, Tyr, Trp
[0111] Residues Pro, Gly and Cys are parenthesized because they can
have special conformational roles. Cys participates in formation of
disulfide bonds. Gly imparts flexibility to the chain. Pro imparts
rigidity to the chain and disrupts a helices. These residues may be
essential in certain regions of the polypeptide, but substitutable
elsewhere.
[0112] Two regulatory DNA sequences (e.g., promoters) are
"substantially homologous" if they have substantially the same
regulatory effect as a result of a substantial identity in
nucleotide sequence. Typically, "substantially homologous"
sequences are at least 50%, more preferably at least 80%,
identical, at least in regions known to be involved in the desired
regulation. Most preferably, no more than five bases are
different.
[0113] For the purposes of the appended claims, the term "chimeric
protein" refers to a protein which is not identical in sequence to
either of two patental proteins A and B, but which, when its
sequence is aligned with the sequences of A and B, can be seen to
borrow features (identically or conservatively) from both parental
proteins.
[0114] The term "autocrine cell", as used herein, refers to a cell
which produces a substance which can stimulate the pheromone system
pathway of that cell. Wild-type .alpha. and a cells are not
autocrine. However a yeast cell which produces both .alpha.-factor
and .alpha.-factor receptor, or both a-factor and a-factor
receptor, in functional form, is autocrine. By extension, yeast
cells which produce a peptide which is being screened for the
ability to activate the pheromone system pathway (e.g., by
activating a G protein-coupled receptor) are called "autocrine
cells", though it might be more precise to call them "putative
autocrine cells". Of course, in a library of such cells, in which a
multitude of different peptides are produced, it is likely that one
or more of the cells will be "autocrine" in the stricter sense of
the term.
[0115] Farnesyltransferases
[0116] The activity of yeast a-factor requires its farnesylation
(mediated by protein farnesyltransferase, comprised of Ram1p and
Ram2p), proteolytic cleavage of the C-terminal 3 amino acids of the
primary translation product (mediated by an as yet unidentified
enzyme), and carboxymethylation of the C-terminal cysteine
(mediated by Ste14p). The yeast and mammalian farnesyltransferases
are structurally and functionally similar (Gomez R et al., Biochem.
J. 289:25-31, 1993; Kohl N E et al., J. Biol. Chem. 266:18884-8,
1991). Sequence homologies exist between the genes encoding the
.alpha. and .beta. subunits of the yeast farnesyltransferase (RAM2
and RAM1, respectively) and the genes encoding the .alpha. and
.beta. subunits of the mammalian farnesytransferase (Kohl N E et
al., J. Biol. Chem. 266:18884-8, 1991; Chen W J et al., Cell
66:327-34, 1991). It has been observed that the .beta. subunit of
mammalian farnesytransferase and Ram1p are 37% identical in amino
acid sequence (Chen W J et al., Cell 66:327-34, 1991).
[0117] The importance of a screen for inhibitors of farnesyl
transferase is suggested by the facts that mammalian p21ras, a
preeminent regulator of the growth and differentiation of mammalian
cells that is involved in a variety of cancers, is a substrate for
the farnesyltransferase and that farnesylation of p21ras is
required for its activity. In fact, a synthetic organic inhibitor
of farnesyl protein transferase has been shown to selectively
inhibit ras-dependent cell transformation (Kohl et al., Science
260, 1934 (1993). Of the two subunits of farnesyltransferase, the
.beta. subunit is a more attractive target for inhibitors, since it
is apparently dedicated to farnesylation. The .alpha. subunit, in
contrast, is shared by geranyl-geranyltransferase I, an enzyme
involved in the modification of the G.gamma. subunits of
heterotrimeric G proteins and small molecular weight G proteins of
the Rho/Rac family. While the .beta. subunit is dedicated to
farnesylation, the mammalian farnesyltransferase has a variety of
substrates in addition to p21ras. The effect of inhibitors of the
.beta. subunit on the farnesylation of these other substrates,
e.g., lamin proteins, transducin-.gamma. and rhodopsin kinase, will
be considered in the design and use of potential
farnesyltransferase inhibitors.
[0118] It has not yet been demonstrated that the homologous
mammalian gene will functionally substitute for yeast Ram1p,
however, this can be formally tested using ram1 mutants and a
vector expressing the mammalian gene encoding the .beta. subunit of
the farnesyltransferase. If the mammalian .beta. subunit can
function in place of Ram1p, test cells will be both viable (as a
result of farnesylation of Ras) and competent for mating (as a
result of farnesylation of a-factor).
[0119] If the mammalian gene encoding the .beta. subunit of
farnesyltransferase complements ram1, yeast would provide a test
system for the discovery of potential inhibitors of mammalian
farnesyltransferase. Specifically, MATa yeast tester cells could be
exploited that: 1. carry the gene for the .beta. subunit of
mammalian farnesyltransferase in lieu of RAM1; 2. carry the cam
mutation that renders the strains resistant to loss of Ras function
in the presence of cAMP; 3. respond to a-factor which they export
by virtue of heterologous expression of Ste3p; 4. respond to
autocrine a-factor such that they cannot grow on media containing
galactose. The latter characteristic will require expression of
GAL1 under the control of a pheromone-responsive promoter and cells
engineered to contain mutated GAL7 or GAL10 genes. Expression of
GAL1 is toxic in the presence of galactose in strains which contain
mutations in either the GAL7 or GAL10 genes. Signaling through the
pheromone response pathway would render cells so engineered
galactose-sensitive. Exposure of such strains to compounds which
inhibit the .beta. subunit of farnesyltransferase will confer upon
these cells the ability to grow on media containing galactose and
cAMP.
[0120] If the mammalian gene encoding the .beta. subunit of
farnesyltransferase (and all modified versions of the gene) fails
to complement ram1, we may use the wild-type Ram1p as a surrogate
target for potential effectors of mammalian farrnesyltransferase.
Specifically, we will use as tester cells MATa yeast strains that:
1. carry the cam mutation that renders the strains resistent to
loss of RAS function in the presence of cAMP; 2. respond to
a-factor which they export by virtue of heterologous expression of
Ste3p; 3. respond to autocrine a-factor such that they cannot grow
on media containing galactose. Exposure of such strains to
compounds which inhibit the .beta. subunit of farnesytransferase
will confer upon these cells the ability to grow on media
containing galactose and cAMP.
[0121] In the strategies outlined above, it is desirable to
discriminate inhibitors of farnesytransferase from compounds that
either directly block the negative response to a-factor, e.g. by
interfering with the interaction of the Ste4-Ste18 complex with its
effector, or by blocking the production of a-factor by a mechanism
that does not involve farnesyltransferase. Controls would identify
such false positives. Candidate agents will be tested on a MATa
strain that is engineered to secrete .alpha.-factor and to respond
to the secreted a-factor by failing to grow on galactose-containing
media, as in the negative selection scheme outlined above. The
strain will express wild type Ram1p. Any agent that enables these
cells to grow on media containing galactose and cAMP will not be
acting as an inhibitor of farnesyltransferase.
[0122] Candidate compounds which pass the foregoing test may act by
targeting Ste14p, Ste6p, or other proteins involved in the
maturation and export of a-factor, rather than farnesyltransferase.
(Note, however, that compounds that inhibit processes critical to
cell survival will not give rise to false positives. For example,
since the protease responsible for the endoproteolytic removal of
the C-terminal tripeptide of the a-factor precursor likely
participates in the processing of Gg and members of the Rho/Rac
family of proteins, inhibitors of this enzyme may not permit growth
of the tester cells). Of the proteins involved in the production of
a-factor, only the farnesyltransferase is also a major determinant
of RAS function. Due to this effect, ram1 mutants are defective for
growth at 30.degree. C. and completely unable to grow at 37 (He B
et al., Proc Natl Acad Sci 88:11373-7, 1991). Tester cells
(described above) can be grown in the presence of a candidate
inhibitor on galactose-containing media.+-.cAMP. If the test
compound inhibits farnesyltransferase, cells will be capable of
growth on galactose+cAMP but not on galactose in the absence of
cAMP. This difference may be most obvious at 37.degree.. If, on the
other hand, the test compound inhibits other proteins involved in
a-factor production, cells will grow on galactose-containing media
regardless of the presence or absence of cAMP.
[0123] Compounds which pass the above tests are likely inhibitors
of farnesyltransferase. This can be confirmed and their potencies
determined with direct in vitro enzyme assays. Note that the
strategies outlined will identify farnesyltransferase inhibitors
which affect Ram1p. Agents which block Ram2p would likely fail to
grow under all conditions. Indeed, ram2 null mutations are lethal
(He B et al., Proc Natl Acad Sci 88:11373-7, 1991), perhaps due to
the fact that Ram2p also functions as a component of
geranylgeranyltransferase I.
[0124] Carboxymethyltransferases
[0125] In yeast, methylation of the C-terminal amino acid of
a-factor, Ras proteins, and presumably Rho/Rac proteins is
catalyzed by Ste14p. Although MATa ste14 mutants are unable to
mate, reflecting the requirement of carboxymethylation for the
activity of a-factor, ste14 disruptions are not lethal and do not
affect the rate of cell proliferation. Carboxymethylation appears
to be dispensible for the function of Ras proteins and Ste18p (the
yeast homologue of the G.gamma. subunit). Although Ras function in
yeast can apparently tolerate the absence of carboxymethyl
modification, it is nonetheless possible that inhibitors of
mammalian methyltransferases could alter the activity of mammalian
p21ras.
[0126] It could be determined if yeast ste14 mutations can be
complemented by the homologous mammalian gene, or a modified
version of it. One would use an episomal vector to express the
mammalian gene encoding the methyltransferase in yeast that are
genotypically ste14. The strain would be a modified MATa strain
that expresses the a-factor receptor in lieu of the normal a-factor
receptor and that contains an integrated fus1-HIS3 construct, so
that the a-factor secreted by the cell confers autocrine growth on
histidine-deficient media. If the mammalian methyltransferase can
function in place of Ste14p, the tester cells will be capable of
mating. That is, the mammalian methyltransferase will permit
synthesis of active a-factor in ste14 mutants.
[0127] If the mammalian gene encoding the methyltransferase will
complement ste14, tester strains can be constructed to test for
potential inhibitors of mammalian methyltransferase. In one
embodiment, tester MATa yeast strains will: 1. carry a mammalian
carboxymethyltransferase gene in lieu of STE14; 2. respond, to
a-factor which they export by virtue of heterologous expression of
Ste3p; 3. respond to autocrine a-factor such that they cannot grow
on media containing galactose as in the negative GAL1 selection
scheme outlined above. Exposure of such strains to compounds which
inhibit the methyltransferase will confer upon these cells the
ability to grow on media containing galactose.
[0128] It is desirable to discriminate inhibitors of
carboxymethyltransferase activity from compounds that either
directly block the negative response to a-factor, e.g. by
interfering with the interaction of the Ste4-Ste18 complex with its
effector, or block the production of a-factor by a mechanism that
does not involve methyltransferase. The following control
experiments will identify such false positives. Candidate
inhibitors will be tested on a MATa strain that is engineered to
secrete a-factor and to respond to the secreted a-factor by failing
to grow on galactose-containing media. Any agent that enables these
cells to grow on media containing galactose will be not be acting
as an inhibitor of carboxymethyltransferase. Candidate compounds
which pass the foregoing test may be targetting the
carboxymethyltransferase, farnesyltransferase, Ste6p, or other
proteins involved in the maturation and export of a-factor. In
order to discriminate the target of the compounds, a combination of
in vitro biochemical and in vivo genetic assays can be applied:
both the carboxymethyltransferase and the farnesyltransferase can
be assayed in vitro to test the effect of the candidate agent.
Furthermore, if the target is Ste14p its overexpression on
high-copy plasmids should confer resistance to the effect of the
compound in vivo.
[0129] Proteases
[0130] Mature yeast .alpha.-factor is a thirteen amino acid peptide
that is derived from a polyprotein precursor in much the same
manner as mature mammalian melanocyte-stimulating hormone (MSH) or
calcitonin are derived from precursor polyproteins. Two genes in
the yeast genome encode prepro-.alpha.-factor, MF.alpha.1 and
MF.alpha.2. MF.alpha.1 encodes a precursor polypeptide containing
four copies of mature .alpha.-factor embedded in a polypeptide of
the following structure: hydrophobic pre-sequence/hydrophilic
pro-sequence/.alpha.-factor/.alpha.-factor/.alph-
a.-factor/.alpha.-factor. MF.alpha.2 encodes a polyprotein
precursor of a similar structure containing only two copies of
mature .alpha.-factor.
[0131] Pre-pro-.alpha.-factor is synthesized in the cytoplasm and
is then transported from the cytoplasm to the endoplasmic reticulum
and then to the Golgi along the classical secretory pathway of S.
cerevisiae. The signal sequence of prepro-.alpha.-factor is cleaved
during transit into the ER by signal peptidase and
asparagine-linked oligosaccharides are added (in the ER) and
modified (in the Golgi) on the pro-segment of the precursor as it
transits the secretory pathway. Once in the Golgi, three distinct
proteolytic processing events occur. First, the Kex2 protease
cleaves at dibasic residues (-KR-) near the amino terminus of each
.alpha.-factor repeat. Kex2 is homologous to the subtilisin-like
endoproteases PC2 and PC1/PC3 involved in prohormone processing in
mammalian cells (Smeekens and Steiner 1990; Nakayama et al. 1991).
Additional mammalian Kex2-like processing endoproteases include
PACE, isolated from a human hepatoma, PC4, expressed in testicular
germ cells and PC6, a candidate protease for the processing of
gastrointestinal peptides (Barr et al. 1991; Nakayama et al. 1992;
Nakagawa et al. 1993). It appears that Kex2-like proteins comprise
a large family of tissue-specific endoproteases in mammalian
cells.
[0132] Once Kex2 has released the immature .alpha.-factor peptides,
two additional proteases act to complete processing. Kex1 is a
specific carboxypeptidase that removes the carboxy-terminal-KR
remaining after cleavage by Kex2. Like its mammalian counterparts
carboxypeptidases B and E, Kex1 is highly specific for peptide
substrates with carboxy-terminal basic residues. The final
proteolytic processing event that occurs is the removal of the
spacer dipeptides at the amino terminus of each pro-.alpha.-factor
peptide. This is accomplished by the product of the STE13 gene,
dipeptidyl aminopeptidase A. This enzyme is a type IV dipeptidyl
aminopeptidase: it is capable of cleaving on the carboxyl side of
either -x-A- or -x-P- sites in vitro.
[0133] Other type IV dipeptidyl aminopeptidases are believed to be
active in the processing of a variety of pre-peptides in animal
cells (Kreil 1990). In addition, functional similarity has been
proved between yeast Kex1 and Kex2 and their mammalian
counter-parts in that both yeast enzymes will proteolytically
cleave endogenous precursors when expressed in mammalian cells
deficient in the native enzyme (Thomas et al. 1988, 1990). It
appears likely, then, that mammalian homologs of the yeast
proteases Kex1, Kex2 and Ste13p, when expressed in yeast, will
function to process a synthetic .alpha.-factor pheromone precursor
bearing appropriate cleavage sites. Human proteases that may so
function in yeast include PC2 and PC1/PC3 (or other Kex2 homologs),
carboxypeptidases B and E (Kex1 homologs) and type IV dipeptidyl
aminopeptidases (Ste13p homologs).
[0134] Yeast would provide a facile assay system for the discovery
of inhibitors of proteases able to process synthetic
.alpha.-factor. The yeast could be engineered to express both the
potential inhibitor and the exogenous protease, and, preferably,
not the latter's yeast cognate.
[0135] Furthermore, this means of exploiting yeast pheromone
processing to identify protease inhibitors can be expanded to
encompass any protease that can be expressed to function in yeast,
provided an appropriate cleavage recognition site is included in a
synthetic .alpha.-factor precursor. In the latter approach, novel
proteolytic activities will be added to yeast; these enzymes will
substitute for proteases in the .alpha.-factor maturation pathway
but will not be "catalytic homologues" of Kex1, Kex2 or Ste13p.
Production of mature .alpha.-factor will become dependent on the
activity of the novel protease through removal of the recognition
site(s) for a selected yeast enzyme from a synthetic MF.alpha. gene
and insertion of the recognition sequence for the novel
protease(s).
[0136] Enzymes for which inhibitors could be identified via this
strategy include, by way of example, HIV-1 protease or other
virally encoded proteases involved in the maturation of infectious
particles; neutrophil elastase believed to be involved in pulmonary
disease and inflammatory bowel disease; Factor Xa involved in
thrombin processing and clotting disorders; and CD26, a dipeptidyl
peptidase IV and putatively the second receptor for HIV-1 on
CD4.sup.+ cells. In addition, metalloproteinases (e.g. collagenase)
and serine proteases involved in tissue invasion by tumor cells and
in metastasis would be suitable therapeutic targets. In support of
this, it has been demonstrated that administration of
tissue-derived inhibitors of metalloproteinases results in
decreased metastasis in animal models (Schultz et al. 1988).
Collagenases have also been implicated in the destruction of
connective tissue which accompanies inflammatory processes like
rheumatoid arthritis.
[0137] The use of the present invention to screen for modulators of
prohormone convertase PCi is described in Example 8.
[0138] Exogenous ABC Transporters
[0139] The majority of proteins destined for transport to the
extracellular environment proceed through a secretory pathway that
includes translation initiation in the cytoplasm, transport to the
lumen of the endoplasmic reticulum, passage through the Golgi to
secretory vesicles and subsequent exit from cells. Other proteins
leave the cell by an alternative mechanism, which involves
mediation by an "ABC transporter". The ABC transporters form a
family of evolutionarily conserved proteins, share a similar
overall structure, and function in the transport of large and small
molecules across cellular membranes (Higgins 1992). The
characteristic component of members of this protein family is a
highly conserved sequence that binds ATP (Higgins et al., 1986;
Hyde et al. 1990); these intrinsic membrane proteins are ATPases,
deriving energy from the hydrolysis of that nucleotide to effect
the transport of molecules. This family includes over 50
prokaryotic and eukaryotic proteins: transporters of amino acids,
sugars, oligosaccharides, ions, heavy metals, peptides, or other
proteins belong to this superfamily. Representative transmembrane
transporters are included in Table 1. Typically, ABC transporters
use the energy of ATP hydrolysis to pump substrate across a cell
membrane against a concentration gradient. Some import substrate,
others export it. See Higgins, Ann. Rev. Cell, Biol., 8:67-113
(1992).
[0140] The prototypical structure of an ABC transporter includes
four membrane-associated domains: two hydrophobic, putative
membrane-spanning sequences, each predicted to traverse the
membrane six times, and two nucleotide binding domains that couple
ATP hydrolysis to transport. In prokaryotes, the domains of an ABC
transporter are often present on separate polypeptides. Various
permutations of domain fusions have been described: the E. coli
iron hydroxamate transporter contains the two membrane-spanning
domains in a single polypeptide and the ribose transporter of the
same organism bears two nucleotide-binding domains on one molecule.
The major histocompatibility complex (MHC) peptide transporter is
composed of two polypeptides, Tap1 and Tap2. The N-terminus of each
protein contains a hydrophobic membrane-spanning domain while the
C-terminus contains an ATP-binding sequence. Together Tap1 and Tap2
form a functional complex. The heavy metal tolerance protein, HMT1,
expressed in the fission yeast Schizosaccharomyces pombe, consists
of a polypeptide containing a single hydrophobic domain and a
C-terminal ATP-binding sequence (Ortiz et al. 1992). It may be that
the HMT1 transporter functions as a homodimer. The Saccharomyces
cerevisiae Ste6 a-factor transporter is expressed as a single
polypeptide containing two membrane-spanning domains and two
nucleotide-binding domains. When Ste6 is expressed as two
half-molecules, the protein complex which apparently forms retains
function at a level greater than 50% that of the wild type, single
polypeptide (Berkower and Michaels 1991). In other eukaryotic ABC
transporters, including Mdr1, CFTR and MRP, the four domains are
also contained within a single polypeptide. Thus, the ABC
transporter may be a single multidomain polypeptide, or it may
comprise two or more polypeptides, each providing one or more
domains.
[0141] In general, transporters contain six transmembrane segments
per each hydrophobic domain, for a total of twelve segments. The
minimum number of transmembrane segments required for formation of
a translocation complex appears to be 10. Thus the histidine
transporter of S. typhimurium lacks an N-terminal transmembrane
segment from each of its hydrophophic domains and therefore
contains five transmembrane segments per domain (Higgins et al.,
Nature 298, 723-727 (1982). The MalF protein of the E. coli maltose
transporter contains an N-terminal extension of hydrophobic
sequence which bears two additional transmembrane segments,
bringing the total for this hydrophobic domain to 8 (Overduin et
al. 1988). The N-terminal extension can be deleted, however,
without loss of function of this transporter (Ehrmann et al. 1990).
Although the number of segments required for formation of a
functional translocator is suggested by these studies, there exists
no data on the precise structure of the transmembrane segments
themselves. These sequences are assumed to have an .alpha.-helical
form, but this has not been proven and the structure of the entire
translocation complex within the plasma membrane remains to be
elucidated.
[0142] In order to span the lipid bilayer, a minimum of 20 amino
acids is required and sequences believed to form transmembrane
segments have been identified using hydrophobicity scales.
Hydrophobicity scales assign values to individual amino acid
residues indicating the degree of hydrophobicity of each molecule
(Kyte and Doolittle 1982; Engleman et al. 1986). These values are
based on experimental data (solubility measurements of amino acids
in various solvents, analysis of side chains within soluble
proteins) and theoretical considerations, and allow prediction of
secondary structure in novel sequence with reasonable accuracy.
Analysis using hydrophobicity measurements indicates those
stretches of a protein sequence which have the hydrophobic
properties consistent with a transmembrane helix.
[0143] With a few exceptions, there is little or no significant
amino acid sequence similarity between the transmembrane domains of
two different transporters. This lack of sequence similarity is not
inconsistent with the apparent function of these hydrophobic
domains. While these residues must be capable of forming the
hydrophobic .alpha.-helical structures believed to transverse the
plasma membrane, many amino acid residues are hydrophobic and can
contribute to the formation of an .alpha.-helix.
[0144] Considerable, if as yet inexplicable, sequence similarity
has been detected in comparisons of the transmembrane domains of
the yeast STE6, human MDR and E. coli HlyB hemolysin transporters
[Gros et al., Cell 47, 371 (1986); McGrath and Varchavsky, Nature
340, 400 (1989); Kuchler et al., EMBO J. 8, 3973 (1989)]. Other
sequence similarities can be explained by gene duplication, as in
the case of the transmembrane domains of rodent P-glyco-proteins
(Endicott et al. 1991). The transmembrane domain of the histidine
transporter of S. typhimurium bears homology to that of the
octopine uptake system of Agrobacterium tumefaciens, the latter two
transporters translocate chemically similar substrates (Valdiva et
al. 1991).
[0145] Study of mutant transport proteins has pointed to a role for
the transmembrane sequences in the recognition of substrate. Thus
maltose transporters in E. coli which gain the ability to
translocate p-nitrophenyl-.alpha.-maltoside bear mutations in the
transmembrane domain (Reyes et al. 1986). A mutation in
transmembrane segment 11 of MDR has been shown to change the
substrate specificity of that transporter (Gros et al. 1991) and
mutation of charged residues in the transmembrane domain of CFTR
changes its ion selectivity (Anderson et al. 1991).
[0146] Some aspects of the involvement of extramembrane loop
sequences in transport function are being elucidated. In a number
of bacterial transporters a short conserved motif is present on the
cytoplasmic loop which connects transmembrane segments 4 and 5
[Dassa and Hofnung (1985)]. It has been hypothesized that this
sequence interacts with the ATP-binding domains of these transport
proteins; mutation of this conserved sequence will abolish
transport function (Dassa 1990). Cytoplasmic loops may also be
involved in substrate recognition. Thus the sequences following
transmembrane segments 7 and 12 of the yeast a-factor transporter
resemble sequences in the a-factor receptor, Ste3p, and may
interact with the pheromone substrate (Kuchler et al. 1989). In
fact, mutations in the cytoplasmic loops are known to alter the
substrate specificity of a given transporter. The G185V mutation of
human MDR, located in the loop between transmembrane segments 2 and
3, alters the interaction of that transporter with vinblastine and
colchicine (Choi et al. 1988).
[0147] The ATP-binding domains are about 200 amino acids long, and
domains from different transporters typically have a sequence
identity of 30-50%. The conserved sequences include the "Walker
motifs" which are associated with many nucleotide binding proteins.
Walker, et al., EMBO J. 1:945-951 (1982). Sequence conservation
extends over the length of the ATP-binding domain, not being
limited to the Walker motifs. Furthermore, the ATP-binding domains
of a single transporter exhibit greater sequence identity to one
another than to the domains from two different transporters. Not
all proteins containing a conserved ATP-binding domain are involved
in transport, however. The cytoplasmic enzyme UvrA functions in DNA
repair and the EF-3 protein of yeast is an elongation factor. Yet
both proteins contain ATP-binding cassettes identifiable by
sequence comparison.
[0148] ATP-binding domains are highly hydrophilic and, in the case
of transporters, appear to reside at the cytoplasmic face of the
membrane, anchored there via an association with the
membrane-spanning domain of these proteins. The points of
interaction between the transmembrane and ATP-binding domains have
not been experimentally determined. Models of the structure of the
nucleotide binding domain indicate that loop sequences may extend
from the core of the structure to interface with the hydrophilic
sequences which transverse the membrane (Hyde et al. 1990; Mimura
et al. 1991). The two structural models, one based on adenylate
cyclase and the other on ras p21 structure, predict a core
nucleotide binding fold composed of five .beta.-sheets with the
Walker A motif (a glycine-rich loop) positioned to interact with
ATP during hydrolysis. In addition, loop structures (two loops in
one model, one large loop in the other) are predicted to extend
from the core to couple the ATP-binding domain to other domains of
the transporter. The coupling sequences transmit, most likely
through conformational change, the energy of ATP hydrolysis to
those portions of the molecule which are involved in transport.
[0149] Ste6 function is required for mating but the protein is not
necessary for yeast survival (Wilson and Herskowiz 1984; Kuchler et
al. 1989; McGrath and Varshavsky 1989). Ste6 is structurally
homologous to the mammalian MDRs. Furthermore, it has been
demonstrated that two mammalian MDR proteins, murine Mdr3 and human
Mdr1, will substitute functionally for the yeast transporter in
cells deleted for STE6 (Raymond et al. 1992; Kuchler and Thorner
1992). Yeast strains deleted for STE6 serve as a starting point for
the design of screens to discover compounds that modulate the
function of exogenous ABC transporters.
[0150] Two different yeast screens can be used to identify
modulators of ABC transporter function. In the first instance, a
mammalian protein that transports a-factor will serve as a target
for potential inhibitors of transporter function. Thus, a yeast
strain will be engineered to express a functional transporter, e.g.
mammalian MDR1, which substitutes for the yeast Ste6 protein in the
transport of a-factor. Furthermore, this strain will be engineered
to respond in autocrine fashion to a-factor: e.g., so that the
cells will be unable to grow on media containing galactose. This
negative selection will depend on the expression of the GAL1 gene
under the control of a pheromone-responsive promoter in a strain
background which includes mutated versions of the GAL7 or GAL10
genes. Expression of GAL1 in the presence of galactose in such a
strain background is toxic to cells. In the absence of a-factor
transport, signaling down the pheromone response pathway would
cease as would the consequent expression of the toxic gene. Cell
growth in the presence of a test compound, or upon expression of a
specific random peptide, would signal inhibition of transport
function and the identification of a potential therapeutic.
[0151] In addition to inhibitors of MDR, compounds may be
identified which interfere with the interaction of a-factor with
the a-factor receptor. Such compounds can be discriminated by their
inhibition of a-factor-induced growth arrest in a wild type
Mat.alpha. strain. Compounds may also impact at other points along
the pheromone response pathway to inhibit signaling and these
compounds will prevent signal transduction in a wild type
Mat.alpha. strain.
[0152] In a second screen, a mutant heterologous transporter (e.g.,
mutant CFTR) that is initially incapable of transporting a-factor
or an a-factor-like peptide can be expressed in autocrine yeast
deleted for endogenous Ste6. The cells will be capable of an
autocrine response to the a-factor which those cells produce. Thus
a pheromone-responsive promoter will control expression of a gene
that confers an ability to grow in selective media. Such cells will
permit identification of compounds which correct defects in the
transporter and permit it to function in the export of pheromone
analogues to the extracellular space. In this way, therapeutic
peptides or other classes of chemical compounds could be identified
which stabilize a mutant protein and allow normal processing,
transport, localization to the plasma membrane and function. This
strategy, if successful, may eliminate the need to "replace" some
mutant genes with normal sequence, as envisioned in gene therapies,
by recovering the function of mutant proteins through the
correction of processing and/or localization defects.
[0153] In addition to "activators" of the mutant transporter,
compounds may also be identified which are capable of initiating
signalling from the a-factor receptor in the absence of transport
by the endogenously expressed pheromone. These compounds will be
distinguished by their ability to cause growth arrest in a wild
type Mat.alpha. strain. Compounds may also impact at other points
along the pheromone pathway and can be discerned via an ability to
initiate signalling in a wild type Mat.alpha. strain in the absence
of a-factor.
[0154] In a preferred embodiment, the exogenous protein produced by
the yeast cells is one of the exogenous ABC transporters listed in
Table 1.
[0155] Exoqenous G Protein Coupled Receptors
[0156] In some instances, for a drug to cure a disease or alleviate
its symptoms, the drug must be delivered to the appropriate cells,
and trigger the proper "switches." The cellular switches are known
as "receptors." Hormones, growth factors, neurotransmitters and
many other biomolecules normally act through interaction with
specific cellular receptors. Drugs may activate or block particular
receptors to achieve a desired pharmaceutical effect. Cell surface
receptors mediate the transduction of an "external" signal (the
binding of a ligand to the receptor) into an "internal" signal (the
modulation of a cytoplasmic metabolic pathway).
[0157] In many cases, transduction is accomplished by the following
signaling cascade:
[0158] An agonist (the ligand) binds to a specific protein (the
receptor) on the cell surface.
[0159] As a result of the ligand binding, the receptor undergoes an
allosteric change which activates a transducing protein in the cell
membrane.
[0160] The transducing protein activates, within the cell,
production of so-called "second messenger molecules."
[0161] The second messenger molecules activate certain regulatory
proteins within the cell that have the potential to "switch on" or
"off" specific genes or alter some metabolic process.
[0162] This series of events is coupled in a specific fashion for
each possible cellular response. The response to a specific ligand
may depend upon which receptor a cell expresses. For instance, the
response to adrenalin in cells expressing .alpha.-adrenergic
receptors may be the opposite of the response in cells expressing
.beta.-adrenergic receptors
[0163] The above "cascade" is idealized, and variations on this
theme occur. For example, a receptor may act as its own transducing
protein, or a transducing protein may act directly on an
intracellular target without mediation by a "second messenger".
[0164] One family of signal transduction cascades found in
eukaryotic cells utilizes heterotrimeric "G proteins." Many
different G proteins are known to interact with receptors. G
protein signaling systems include three components: the receptor
itself, a GTP-binding protein (G protein), and an intracellular
target protein.
[0165] The cell membrane acts as a switchboard. Messages arriving
through different receptors can produce a single effect if the
receptors act on the same type of G protein. On the other hand,
signals activating a single receptor can produce more than one
effect if the receptor acts on different kinds of G proteins, or if
the G proteins can act on different effectors.
[0166] In their resting state, the G proteins, which consist of
alpha (.alpha.), beta (.beta.) and gamma (.gamma.) subunits, are
complexed with the nucleotide guanosine diphosphate (GDP) and are
in contact with receptors. When a hormone or other first messenger
binds to receptor, the receptor changes conformation and this
alters its interaction with the G protein. This spurs the .alpha.
subunit to release GDP, and the more abundant nucleotide guanosine
tri-phosphate (GTP), replaces it, activating the G protein. The G
protein then dissociates to separate the .alpha. subunit from the
still complexed beta and gamma subunits. Either the G.alpha.
subunit, or the G.beta..gamma. complex, depending on the pathway,
interacts with an effector. The effector (which is often an enzyme)
in turn converts an inactive precursor molecule into an active
"second messenger," which may diffuse through the cytoplasm,
triggering a metabolic cascade. After a few seconds, the G.alpha.
converts the GTP to GDP, thereby inactivating itself. The
inactivated G.alpha. may then reassociate with the G.beta..gamma.
complex.
[0167] Hundreds, if not thousands, of receptors convey messages it
through heterotrimeric G proteins, of which at least 17 distinct
forms have been isolated. Although the greatest variability has
been seen in the a subunit, several different .beta. and .gamma.
structures have been reported. There are, additionally, several
different G protein-dependent effectors.
[0168] Most G protein-coupled receptors are comprised of a single
protein chain that is threaded through the plasma membrane seven
times. Such receptors are often referred to as seven-transmembrane
receptors (STRs). More than a hundred different STRs have been
found, including many distinct receptors that bind the same ligand,
and there are likely many more STRs awaiting discovery.
[0169] In addition, STRs have been identified for which the natural
ligands are unknown; these receptors are termed "orphan" G
protein-coupled receptors. Examples include receptors cloned by
Neote et al. Cell 72, 415 (1993); Kouba et al. FEBS Lett. 321, 173
(1993); Birkenbach et al. J. Virol. 67, 2209 (1993).
[0170] The "exogenous G protein-coupled receptors" of the present
invention may be any G protein-coupled receptor which is exogenous
to the wild-type yeast cell which is to be genetically engineered
for the purpose of the present invention. This receptor may be a
plant or animal cell receptor. Screening for binding to plant cell
receptors may be useful in the development of, e.g., herbicides. In
the case of an animal receptor, it may be of invertebrate or
vertebrate origin. If an invertebrate receptor, an insect receptor
is preferred, and would facilitate development of insecticides. The
receptor may also be a vertebrate, more preferably a mammalian,
still more preferably a human, receptor. The exogenous receptor is
also preferably a seven transmembrane segment receptor.
[0171] Suitable receptors include, but are not limited to,
dopaminergic, muscarinic cholinergic, .alpha.-adrenergic,
.beta.-adrenergic, opioid (including delta and mu), cannabinoid,
serotoninergic, and GABAergic receptors. Other suitable receptors
are listed in Table 2. The term "receptor," as used herein,
encompasses both naturally occurring and mutant receptors.
[0172] Many of these G protein-coupled receptors, like the yeast a-
and .alpha.-factor receptors, contain seven hydrophobic amino
acid-rich regions which are assumed to lie within the plasma
membrane. Specific human G protein-coupled STRs for which genes
have been isolated and for which expression vectors could be
constructed include those listed in Table 2. Thus, the gene would
be operably linked to a promoter functional in yeast and to a
signal sequence functional in yeast. Suitable promoters include
Ste2, Ste3 and gal10. Suitable signal sequences include those of
Ste2, Ste3 and of other genes which encode proteins secreted by
yeast cells. Preferably, the codons of the gene would be optimized
for expression in yeast. See Hoekema et al., Mol. Cell. Biol.,
7:2914-24 (1987); Sharp, et al., 14:5125-43 (1986).
[0173] The homology of STRs is discussed in Dohlman et al., Ann.
Rev. Biochem., 60:653-88 (1991). When STRs are compared, a distinct
spatial pattern of homology is discernable. The transmembrane
domains are often the most similar, whereas the N- and C-terminal
regions, and the cytoplasmic loop connecting transmembrane segments
V and VI are more divergent.
[0174] The functional significance of different STR regions has
been studied by introducing point mutations (both substitutions and
deletions) and by constructing chimeras of different but related
STRs. Synthetic peptides corresponding to individual segments have
also been tested for activity. Affinity labeling has been used to
identify ligand binding sites.
[0175] It is conceivable that a foreign receptor which is expressed
in yeast will functionally integrate into the yeast membrane, and
there interact with the endogenous yeast G protein. More likely,
either the receptor will need to be modified (e.g., by replacing
its V-VI loop with that of the yeast STE2 or STE3 receptor), or a
compatible G protein should be provided.
[0176] If the wild-type exogenous G protein-coupled receptor cannot
be made functional in yeast, it may be mutated for this purpose. A
comparison would be made of the amino acid sequences of the
exogenous receptor and of the yeast receptors, and regions of high
and low homology identified. Trial mutations would then be made to
distinguish regions involved in ligand or G protein binding, from
those necessary for functional integration in the membrane. The
exogenous receptor would then be mutated in the latter region to
more closely resemble the yeast receptor, until functional
integration was achieved. If this were insufficient to achieve
functionality, mutations would next be made in the regions involved
in G protein binding. Mutations would be made in regions involved
in ligand binding only as a last resort, and then an effort would
be made to preserve ligand binding by making conservative
substitutions whenever possible.
[0177] Preferably, the yeast genome is modified so that it is
unable to produce the endogenous a- and .alpha.-factor receptors in
functional form. Otherwise, a positive assay score might reflect
the ability of a peptide to activate the endogenous G
protein-coupled receptor, and not the receptor of interest.
[0178] G Protein
[0179] When the PSP surrogate is an exogenous G protein-coupled
receptor, the yeast cell must be able to produce a G protein which
is activated by the exogenous receptor, and which can in turn
activate the yeast effector(s). It is possible that the endogenous
yeast G.alpha. subunit (e.g., GPA) will be sufficiently homologous
to the "cognate" G.alpha. subunit which is natively associated with
the exogenous receptor for coupling to occur. More likely, it will
be necessary to genetically engineer the yeast cell to produce a
foreign G.alpha. subunit which can properly interact with the
exogenous receptor. For example, the G.alpha. subunit of the yeast
G protein may be replaced by the G.alpha. subunit natively
associated with the exogenous receptor.
[0180] Dietzel and Kurjan, Cell, 50:1001 (1987) demonstrated that
rat G.alpha.s functionally coupled to the yeast G.beta..gamma.
complex. However, rat G.alpha.i2 complemented only when
substantially overexpressed, while G.alpha.0 did not complement at
all. Kang, et al., Mol. Cell. Biol., 10:2582 (1990). Consequently,
with some foreign G.alpha. subunits, it is not feasible to simply
replace the yeast G.alpha..
[0181] If the exogenous G protein coupled receptor is not
adequately coupled to yeast G.beta..gamma. by the G.alpha. subunit
natively associated with the receptor, the G.alpha. subunit may be
modified to improve coupling. These modifications often will take
the form of mutations which increase the resemblance of the
G.alpha. subunit to the yeast G.alpha. while decreasing its
resemblance to the receptor-associated G.alpha.. For example, a
residue may be changed so as to become identical to the
corresponding yeast G.alpha. residue, or to at least belong to the
same exchange group of that residue. After modification, the
modified G.alpha. subunit might or might not be "substantially
homologous" to the foreign and/or the yeast G.alpha. subunit.
[0182] The modifications are preferably concentrated in regions of
the G.alpha. which are likely to be involved in G.beta..gamma.
binding. In some embodiments, the modifications will take the form
of replacing one or more segments of the receptor-associated
G.alpha. with the corresponding yeast G.alpha. segment (s), thereby
forming a chimeric G.alpha. subunit. (For the purpose of the
appended claims, the term "segment" refers to three or more
consecutive amino acids.) In other embodiments, point mutations may
be sufficient.
[0183] This chimeric G.alpha. subunit will interact with the
exogenous receptor and the yeast G.beta..gamma. complex, thereby
permitting signal transduction. While use of the endogenous yeast
G.beta..gamma. is preferred, if a foreign or chimeric
G.beta..gamma. is capable of transducing the signal to the yeast
effector, it may be used instead.
[0184] G.alpha. Structure
[0185] We will now review information regarding G.alpha. structure
which is relevant to the design of modified G.alpha. subunits.
[0186] In Table 5, part A, the amino terminal 66 residues of GPA1
are aligned with the cognate domains of human G.alpha.s,
G.alpha.i2, G.alpha.i3 and G.alpha.16. In part B, we present
alignment of the amino-terminal 66 residues of GPA1.sub.41-
G.alpha. chimeras. In the GPA.sub.41- G.alpha. hybrids, the amino
terminal 41 residues (derived from GPA1) are identical, end with
the sequence-LEKQRDKNE- and are underlined for emphasis. All
residues following the glutamate (E) residue at position 41 are
contributed by the human G.alpha. subunits, including the consensus
nucleotide binding motif -GxGxxG-. Periods in the sequences
indicate gaps that have been introduced to maximize alignments in
this region. Codon bias is mammalian. For alignments of the entire
coding regions of GPA1 with G.alpha.s, G.alpha.i, and G.alpha.O,
G.alpha.q and G.alpha.z, see Dietzel and Kurjan (1987) and
Lambright, et al. (1994).
[0187] Additional sequence information is provided by Mattera, et
al. (1986), Bray, et al. (1986) and Bray, et al. (1987).
[0188] The sequences are identified as follows: GPA1 (SEQ ID NO:
82); G.alpha.s (SEQ ID NO:83); G.alpha.i2 (SEQ ID NO:84);
G.alpha.i3 (SEQ ID NO:85); G.alpha.16 (SEQ ID NO:86);
GPA41-G.alpha.s (SEQ ID NO:87); GPA41-G.alpha.i2 (SEQ ID NO:88);
GPA41-G.alpha.i3 (SEQ ID NO:89); and GPA41-G.alpha.16 (SEQ ID
NO:90).
[0189] The gene encoding a G protein homolog of S. cerevisiae was
cloned independently by Dietzel and Kurjan (1987) (SCG1) and by
Nakafuku, et al. (1987) (GPA1). Sequence analysis revealed a high
degree of homology between the protein encoded by this gene and
mammalian G.alpha.. GPA1 encodes a protein of 472 amino acids, as
compared with approximately 340-350 a.a. for most mammalian
G.alpha. subunits in four described families, G.alpha.s, G.alpha.i,
G.alpha.q and G.alpha.12/13. Nevertheless, GPA1 shares overall
sequence and structural homology with all G.alpha. proteins
identified to date. The highest overall homology in GPA1 is to the
G.alpha.i family (48% identity, or 65% with conservative
substitutions) and the lowest is to G.alpha.s (33% identity, or 51%
with conservative substitutions) (Nakafuku, et al. 1987).
[0190] The regions of high sequence homology among G.alpha.
subunits are dispersed throughout their primary sequences, with the
regions sharing the highest degree of homology mapping to sequence
that comprises the guanine nucleotide binding/GTPase domain. This
domain is structurally similar to the .alpha..beta. fold of ras
proteins and the protein systhesis elongation factor EF-Tu. This
highly conserved guanine nucleotide-binding domain consists of a
six-stranded .beta. sheet surrounded by a set of five
.alpha.-helices. It is within these .beta. sheets and .alpha.
helices that the highest degree of conservation is observed among
all G.alpha. proteins, including GPA1. The least sequence and
structural homology is found in the intervening loops between the
.beta. sheets and .alpha. helices that define the core GTPase
domain. There are a total of four "intervening loops" or "inserts"
present in all G.alpha. subunits. In the crystal structures
reported to date for the GDP- and GTP.gamma.S-liganded forms of
bovine rod transducin (Noel, et al. 1993; Lambright, et al. 1994),
the loop residues are found to be outside the core GTPase
structure. Functional roles for these loop structures have been
established in only a few instances. A direct role in coupling to
phosphodiesterase-.gamma. has been demonstrated for residues within
inserts 3 and 4 of G.alpha.t (Rarick, et al. 1992; Artemyev, et al.
1992), while a "GAP-like" activity has been ascribed to the largely
.alpha.-helical insert 1 domain of G.alpha.S (Markby, et al.
1993).
[0191] While the amino- and carboxy-termini of G.alpha. subunits do
not share striking homology either at the primary, secondary, or
tertiary levels, there are several generalizations that can be made
about them. First, the amino termini of G.alpha. subunits have been
implicated in the association of G.alpha. with G.beta..gamma.
complexes and in membrane association via N-terminal
myristoylation. In addition, the carboxy-termini have been
implicated in the association of G.alpha..beta..gamma.
heterotrimeric complexes with G protein-coupled receptors
(Sullivan, et al. 1987; West, et al. 1985; Conklin, et al. 1993).
Data in support of these generalizations about the function of the
N-terminus derive from several sources, including both biochemical
and genetic studies.
[0192] As indicated above, there is little if any sequence homology
shared among the amino termini of G.alpha. subunits. The amino
terminal domains of G.alpha. subunits that precede the first
.beta.-sheet (containing the sequence motif -LLLLGAGESG-; see Noel,
et al. (1993) for the numbering of the structural elements of
G.alpha. subunits) vary in length from 41 amino acids (GPA1) to 31
amino acids (G.alpha.t). Most G.alpha. subunits share the consensus
sequence for the addition of myristic acid at their amino termini
(MGxxxS-), although not all G.alpha. subunits that contain this
motif have myristic acid covalently associated with the glycine at
position 2 (speigel, et al. 1991). The role of this
post-translational modification has been inferred from studies in
which the activity of mutant G.alpha. subunits from which the
consensus sequence for myristoylation has been added or deleted has
been assayed (Mumby, et al. 1990; Linder, et al. 1991; Gallego, et
al. 1992). These studies suggest two roles for N-terminal
myristoylation. First, the presence of amino-terminal myristic acid
has in some cases been shown to be required for association of
G.alpha. subunits with the membrane, and second, this modification
has been demonstrated to play a role in modulating the association
of G.alpha. subunits with G.beta..gamma. complexes. The role of
myristoylation of the GPA1 gene product is, at present,
unknown.
[0193] In other biochemical studies aimed at examining the role of
the amino-terminus of G.alpha. in driving the association between
G.alpha. and G.beta..gamma. subunits, proteolytically or
genetically truncated versions of G.alpha. subunits were assayed
for their ability to associate with G.beta..gamma. complexes, bind
guanine nucleotides and/or to activate effector molecules. In all
cases, G.alpha. subunits with truncated amino termini were
deficient in all three functions (Graf, et al. 1992; Journot, et
al. 1990; and Neer, et al. 1988). Slepak, et al. (1993) reported a
mutational analysis of the N-terminal 56 a.a. of mammalian
G.alpha.o expressed in Escherichia coli. Molecules with an apparent
reduced ability to interact with exogenously added mammalian
G.beta..gamma. were identified in the mutant library. As the
authors pointed out, howeve, the assay used to screen the
mutants--the extent of ADP-ribosylation of the mutant G.alpha. by
pertussis toxin--was not a completely satisfactory probe of
interactions between G.alpha. and G.beta..gamma.. Mutations
identified as inhibiting the interaction of the subunits, using
this assay, may still permit the complexing of G.alpha. and
G.beta..gamma. while sterically hindering the ribosylation of
G.alpha. by toxin.
[0194] Genetic studies examined the role of amino-terminal
determinants of G.alpha. in heterotrimer subunit association have
been carried out in both yeast systems using GPA1-mammalian
G.alpha. hybrids (Kang, et al. 1990) and in mammalian systems using
G.alpha.i/G.alpha.s hybrids (Russell and Johnson 1993). In the
former studies, gene fusions, composed of yeast GPA1 and mammalian
G.alpha. sequences were constructed by Kang, et al. (1990) and
assayed for their ability to complement a gpa1 null phenotype
(i.e., constitutive activation of the pheromone response pathway)
in S. cerevisiae. Kang, et al. demonstrated that wild type
mammalian G.alpha.s, G.alpha.i but not G.alpha.o proteins are
competent to associate with yeast G.beta..gamma. and suppress the
gpa1 null phenotype, but only when overexpressed. Fusion proteins
containing the amino-terminal 330 residues of GPA1 sequence linked
to 160, 143, or 142 residues of the mammalian G.alpha.s, G.alpha.i
and G.alpha.o carboxyl-terminal regions, respectively, also coupled
to the yeast mating response pathway when overexpressed on high
copy plasmids with strong inducible (CUP) or constitutive (PGK)
promoters. All three of these hybrid molecules were able to
complement the gpal null mutation in a growth arrest assay, and
were additinally able to inhibit .alpha.-factor responsiveness and
mating in tester strains. These last two observations argue that
hybrid yeast-mammalian G.alpha. subunits are capable of interacting
directly with yeast G.beta..gamma., thereby disrupting the normal
function of the yeast heterotrimer. Fusions containing the amino
terminal domain of G.alpha.s, G.alpha.i or G.alpha.o, however, did
not complement the gpa1 null phenotype, indicating a requirement
for determinants in the amino terminal 330 amino acid residues of
GPA1 for association and sequestration of yeast G.beta..gamma.
complexes. Taken together, these data suggest that determinants in
the amino terminal region of G.alpha. subunits determine not only
the ability to associate with G.beta..gamma. subunits in general,
but also with specific G.beta..gamma. subunits in a
species-restricted manner.
[0195] Hybrid G.alpha.i/G.alpha.s subunits have been assayed in
mammalian expression systems (Russell and Johnson 1993). In these
studies, a large number of chimeric G.alpha. subunits were assayed
for an ability to activate adenylyl cyclase, and therefore,
indirectly, for an ability to interact with G.beta..gamma. (i.e.,
coupling of G.alpha. to G.beta..gamma.=inactive cyclase; uncoupling
of G.alpha. from G.beta..gamma.=active cyclase). From these studies
a complex picture emerged in which determinants in the region
between residues 25 and 96 of the hybrids were found to determine
the state of activation of these alleles as reflected in their
rates of guanine nucleotide exchange and GTP hydrolysis and the
extent to which they activated adenylyl cyclase in vivo. These data
could be interpreted to support the hypothesis that structural
elements in the region between the amino terminal methionine and
the .beta.1 sheet identified in the crystal structure of G.alpha.t
(see Noel, et al. 1993 and Lambright, et al. 1994) are involved in
determining the state of activity of the heterotrimer by (1)
driving association/dissociation between G.alpha. and
G.beta..gamma. subunits; (2) driving GDP/GTP exchange. While there
is no direct evidence provided by these studies to support the idea
that residues in this region of G.alpha. and residues in
G.beta..gamma. subunits contact one another, the data nonetheless
provide a positive indication for the construction of hybrid
G.alpha. subunits that retain function. There is, however, a
negative indicator that derives from this work in that some hybrid
constructs resulted in constitutive activation of the chimeric
proteins (i.e., a loss of receptor-dependent stimulation of
G.alpha..beta..gamma. dissociation and effector activation).
[0196] Construction of Chimeric G.alpha. Subunits
[0197] In designing G.alpha. subunits capable of transmitting, in
yeast, signals originating at mammalian G protein-coupled
receptors, two general desiderata were recognized. First, the
subunits should retain as much of the sequence of the native
mammalian proteins as possible. Second, the level of expression for
the heterologous components should approach, as closely as
possible, the level of their endogenous counterparts. The results
described by King, et al. (1990) for expression of the human
.beta.2-adrenergic receptor and G.alpha.s in yeast, taken together
with negative results obtained by Kang, et al. (1990) with
full-length mammalian G.alpha. subunits other than G.alpha.s, led
us to the following preferences for the development of yeast
strains in which mammalian G protein-coupled receptors could be
linked to the pheromone response pathway.
[0198] 1. Mammalian G.alpha. subunits will be expressed using the
native sequence of each subunit or, alternatively, as minimal gene
fusions with sequences from the amino terminus of GPA1 replacing
the homologous residues from the mammalian G.alpha. subunits.
[0199] 2. Mammalian G.alpha. subunits will be expressed from the
GPA1 promotor either on low copy plasmids or after integration into
the yeast genome as a single copy gene.
[0200] 3. Endogenous G.beta..gamma. subunits will be provided by
the yeast STE4 and STE18 loci.
[0201] Site-Directed Mutagenesis Versus Random Mutagenesis
[0202] There are two general approaches to solving
structure-function problems of the sort presented by attempts to
define the determinants involved in mediating the association of
the subunits that comprise the G protein heterotrimer. The first
approach, discussed above with respect to hybrid constructs, is a
rational one in which specific mutations or alterations are
introduced into a molecule based upon the available experimental
evidence. In a second approach, random mutagenesis techniques,
coupled with selection or screening systems, are used to introduce
large numbers of mutations into a molecule, and that collection of
randomly mutated molecules is then subjected to a selection for the
desired phenotype or a screen in which the desired phenotype can be
observed against a background of undesirable phenotypes. With
random mutagenesis one can mutagenize an entire molecule or one can
proceed by cassette mutagenesis. In the former instance, the entire
coding region of a molecule is mutagenized by one of several
methods (chemical, PCR, doped oligonucleotide synthesis) and that
collection of randomly mutated molecules is subjected to selection
or screening procedures. Random mutagenesis can be applied in this
way in cases where the molecule being studied is relatively small
and there are powerful and stringent selections or screens
available to discriminate between the different classes of mutant
phenotypes that will inevitably arise. In the second approach,
discrete regions of a protein, corresponding either to defined
structural (i.e. a-helices, b-sheets, turns, surface loops) or
functional determinants (e.g., catalytic clefts, binding
determinants, transmembrane segments) are subjected to saturating
or semi-random mutagenesis and these mutagenized cassettes are
re-introduced into the context of the otherwise wild type allele.
Cassette mutagenesis is most useful when there is experimental
evidence available to suggest a particular function for a region of
a molecule and there is a powerful selection and/or screening
approach available to discriminate between interesting and
uninteresting mutants. Cassette mutagenesis is also useful when the
parent molecule is comparatively large and the desire is to map the
functional domains of a molecule by mutagenizing the molecule in a
step-wise fashion, i.e. mutating one linear cassette of residues at
a time and then assaying for function.
[0203] We are applying random mutagenesis in order to further
delineate the determinants involved in G.alpha.-G.beta..gamma.
association. Random mutagenesis may be accomplished by many means,
including:
[0204] 1. PCR mutagenesis, in which the error prone Taq polymerase
is exploited to generate mutant alleles of G.alpha. subunits, which
are assayed directly in yeast for an ability to couple to yeast
G.beta..gamma..
[0205] 2. Chemical mutagenesis, in which expression cassettes
encoding G.alpha. subunits are exposed to mutagens and the protein
products of the mutant sequences are assayed directly in yeast for
an ability to couple to yeast
[0206] 3. Doped synthesis of oligonucleotides encoding portions of
the G.alpha. gene.
[0207] 4. In vivo mutagenesis, in which random mutations are
introducted into the coding region of G.alpha. subunits by passage
through a mutator strain of E. coli, XL1-Red (mutD5 mutS mutT)
(Stratagene, Menasa, Wis.).
[0208] The random mutagenesis may be focused on regions suspected
to be involved in G.alpha.-G.beta..gamma. association as discussed
in the next section. Random mutagenesis approaches are feasible for
two reasons. First, in yeast one has the ability to construct
stringent screens and facile selections (growth vs. death,
transcription vs. lack of transcription) that are not readily
available in mammalian systems. Second, when using yeastit is
possible to screen effienctly through thousands of transformants
rapidly.
[0209] Cassette mutagenesis is immediately suggested by the
observation (see infra) that the GPA.sub.41 hybrids couple to the
pheromone response pathway. This relatively small region of
G.alpha. subunits represents a reasonable target for this type of
mutagenesis. Another region that may be amenable to cassette
mutagenesis is that defining the surface of the switch region of
G.alpha. subunits that is solvent-exposed in the crystal structures
of G.alpha.i1 and transducin. From the data described below, this
surface may contain residues that are in direct contact with yeast
G.beta..gamma. subunits, and may therefore be a reasonable target
for mutagenesis.
[0210] Rational Design of Chimeric G.alpha. Subunits
[0211] Several classes of rationally designed GPA1-mammalian
G.alpha. hybrid subunits have been tested for the ability to couple
to yeast .beta..gamma.. The first, and largest, class of hybrids
are those that encode different lengths of the GPA1 amino terminal
domain in place of the homologous regions of the mammalian G1
subunits. This class of hybrid molecules includes GPA.sub.BamHI,
GPA.sub.41, GPA.sub.ID, and GPA.sub.LW hybrids, described below.
The rationale for constructing these hybrid G.alpha. proteins is
based on results, described above, that bear on the importance of
the amino terminal residues of G.alpha._in mediating interaction
with G.beta..gamma..
[0212] Preferably, the yeast GU subunit is replaced by a chimeric
G.alpha. subunit in which a portion, e.g., at least about 20, more
preferably at least about 40, amino acids, which is substantially
homologous with the corresponding residues of the amino terminus of
the yeast G.alpha., is fused to a sequence substantially homologous
with the main body of a mammalian (or other exogenous) G.alpha..
While 40 amino acids is the suggested starting point, shorter or
longer portions may be tested to determine the minimum length
required for coupling to yeast G.beta..gamma. and the maximum
length compatible with retention of coupling to the exogenous
receptor. It is presently believed that only the final 10 or 20
amino acids at the carboxy terminus of the G.alpha. subunit are
required for interaction with the receptor. GPA.sub.BamHI hybrids.
Kang et al. (1990) described hybrid G.alpha._subunits encoding the
amino terminal 310 residues of GPA1 fused to the carboxyl terminal
160, 143 and 142 residues, respectively, of G.alpha.S, G.alpha.i2,
and G.alpha.o. In all cases examined by Kang et al., the hybrid
proteins were able to complement the growth arrest phenotype of
gpa1 strains. We have confirmed these findings and, in addition,
have constructed and tested hybrids between GPA1 and G.alpha.i3,
G.alpha.q and G.alpha.16 (see Example 11). All hybrids of this type
that have been tested functionally complement the growth arrest
phenotype of gpa1 strains.
[0213] GPA.sub.41 hybrids. The rationale for constructing a minimal
hybrid encoding only 41 amino acids of GPA1 relies upon the
biochemical evidence for the role of the amino-terminus of G.alpha.
subunits discussed above, together with the following observation.
G.beta. and G.gamma. subunits are known to interact via .alpha.
helical domains at their respective amino-termini (Pronin, et al.
1992; Garritsen, et al. 1993). The suggestion that the amino
termini of G.alpha. subunits may form an helical coil and that this
helical coil may be involved in association of G.alpha. with
G.beta..gamma. (Masters, Stroud, and Bourne 1986; Lupas, Lupas and
Stock 1992) leads to the hypothesis that the three subunits of the
G-protein heterotrimer interact with one another reversibly through
the winding and unwinding of their amino-terminal helical regions.
A mechanism of this type has been suggested, as well, from an
analysis of leucine zipper mutants of the GCN4 transcription factor
(Harbury, et al. 1993). The rationale for constructing hybrids like
those described by Kang, et al. (1990), that contain a majority of
yeast sequence and only minimal mammalian sequence, derives from
their ability to function in assays of coupling between G.alpha.
and G.beta..gamma. subunits. However, these chimeras had never been
assayed for an ability to couple to both mammalian G
protein-coupled receptors and yeast G.beta..gamma. subunits, and
hence to reconstitute a hybrid signalling pathway in yeast.
[0214] GPA.sub.41 hybrids that have been constructed and tested
include G.alpha.s, G.alpha.i2, G.alpha.i3, G.alpha.q,
G.alpha.o.sub.a, G.alpha.o.sub.b and G.alpha.16 (see Example 11).
Hybrids of G.alpha.s, G.alpha.i2, G.alpha.i3, and G.alpha.16
functionally complement the growth arrest phenotype of gpa1
strains, while GPA.sub.41 hybrids of G.alpha.o.sub.a and
G.alpha.o.sub.b do not. In addition to being tested in a growth
arrest assay, these constructs have been assayed in the more
sensitive transcriptional assay for activation of a fus1p-HIS3
gene. In both of these assays, the GPA.sub.41-G.alpha.s hybrid
couples less well than the GPA.sub.41-i2, -i3, and -16 hybrids,
while the GPA.sub.41 -o.sub.a and -o.sub.b hyrids do not function
in either assay.
[0215] Several predictive algorithms indicate that the amino
terminal domain up to the highly conserved sequence
motif-LLLLGAGESG- (the first L in this motif is residue 43 in GPA1)
forms a helical structure with amphipathic character. Assuming that
a heptahelical repeat unit, the following hybrids between GPA1 and
GaS can be used to define the number of helical repeats in this
motif necessary for hybrid function:
2 GPA1-7/G.alpha.s8-394 GPA1-14/G.alpha.s15-394
GPA1-21/G.alpha.s22-394 GPA1-28/G.alpha.s29-394
GPA1-35/G.alpha.s36-394 GPA1-42/G.alpha.s43-394
[0216] In this hybrids, the prediction is that the structural
repeat unit in the amino terminal domain up to the tetra-leucine
motif is 7, and that swapping sequences in units of 7 will in
effect amount to a swap of unit turns of turns of the belical
structure that comprises this domain.
[0217] A second group of "double crossover" hybrids of this class
are those that are aligned on the first putative heptad repeat
beginning with residue G11 in GPA1. In these hybrids, helical
repeats are swapped from GPA1 into a GaS backbone one heptad repeat
unit at a time.
3 G.alpha.S1-10/GPA11-17/G.alpha.s18-394
G.alpha.S1-17/GPA18-24/G.alpha.S25-394 G.alpha.S1-17/GPA25-31/G.a-
lpha.S32-394 G.alpha.S1-17/GPA32-38/G.alpha.S39-394
[0218] The gap that is introduced between residues 9 and 10 in the
GaS sequence is to preserve the alignment of the -LLLLGAGE-sequence
motif.
[0219] This class of hybrids can be complemented by cassette
mutagenesis of each heptad repeat followed by screening of these
collections of "heptad" libraries in standard coupling assays.
[0220] A third class of hybrids based on the prediction that the
amino terminus forms a helical domain with a heptahelical repeat
unit are those that effect the overall hydrophobic or hydrophilic
character of the opposing sides of the predicted helical structure
(See Lupas, Stock and Stock). In this model, the .alpha. and d
positions of the heptad repeat abcdefg are found to be conserved
hydrophobic residues that define one face of the helix, while the e
and g positions define the charged face of the helix.
[0221] In this class of hybrids, the sequence of the GaS parent is
maintained except for specific substitutions at one or more of the
following critical residues to render the different helical faces
of GaS more "GPA1-like"
4 K8Q +I-10 E10G Q12E R13S N14D E15P E15F K17L E21R K28Q K32L
V36R
[0222] This collection of single mutations could be screened for
coupling efficiency to yeast G.beta..gamma. and then constructed in
combinations (double and greater if necessary).
[0223] A fourth class of hybrid molecules that span this region of
GPA1-G.alpha. hybrids are those that have junctions between GPA1
and G.alpha. subunits introduced by three primer PCR. In this
approach, the two outside primers are encoded by sequences at the
initiator methionine of GPA1 on the 5' side and at the tetraleucine
motif of G.alpha.S (for example) on the 3' side. A series of
junctional primers spanning different junctional points can be
mixed with the outside primers to make a series of molecules each
with different amounts of GPA1 and GaS sequences, respectively.
[0224] GPA.sub.ID and GPA.sub.LW hybrids. The regions of high
homology among G.alpha. subunits that have been identified by
sequence alignment are interspersed throughout the molecule. The G1
region containing the highly conserved -GSGESGDST- motif is
followed immediately by a region of very low sequence conservation,
the "i1" or insert 1 region. Both sequence and length vary
considerably among the i1 regions of the G.alpha. subunits. By
aligning the sequences of G.alpha. subunits, the conserved regions
bounding the i1 region were identified and two additional classes
of GPA1-G.alpha. hybrids were constructed. The GPA.sub.ID hybrids
encode the amino terminal 102 residues of GPA1 (up to the sequence
-QARKLGIQ-) fused in frame; to mammalian G.alpha. subunits, while
the GPA.sub.LWhybrids encode the amino terminal 244 residues of
GPA1 (up to the sequence--LIHEDIAKA- in GPA1). The reason for
constructing the GPA.sub.ID and GPA.sub.LW hybrids was to test the
hypothesis that the i1 region of GPA1 is required for mediating the
interaction of GPA1 with yeast G.beta..gamma. subunits, for the
stable expression of the hybrid molecules, or for function of the
hybrid molecules. The GPA.sub.ID hybrids contain the amino terminal
domain of GPA1 fused to the i1 domain of mammalian subunits, and
therefore do not contain the GPA1 i1 region, while the GPA.sub.LW
hybrids contain the amino terminal 244 residues of GPA1 including
the entire i1 region (as defined by sequence alignments). Hybrids
of both GPA.sub.ID and GPA.sub.LW classes were constructed for
G.alpha.S, G.alpha.i2, G.alpha.i3, G.alpha.o.sub.a, and G.alpha.16;
none of these hybrids complemented the gpa1 growth arrest
phenotype.
[0225] Subsequent to the construction and testing of the GPA.sub.ID
and GPA.sub.LW classes of hybrids, the crystal structures of
G.alpha..sub.transducin in both the GDP and GTP.gamma.S-liganded
form, and the crystal structure of several G.alpha.i1 variants in
the GTP.gamma.S-liganded and GDP-AlF.sub.4 forms were reported
(Noel et al. 1993; Lambright et al. 1994 and Coleman et al. 1994).
The crystal structures reveal that the ilregion defined by sequence
alignment has a conserved structure that is comprised of six alpha
helices in a rigid array, and that the junctions chosen for the
construction of the GPA.sub.ID and GPA.sub.LW hybrids were not
compatible with conservation of the structural features of the i1
region observed in the crystals. The junction chosen for the
GPA.sub.ID hybrids falls in the center of the long .alpha.A helix;
chimerization of this helix in all likelihood destabilizes it and
the protein structure in general. The same is true of the junction
chosen for the GPA.sub.LW hybrids in which the crossover point
between GPA1 and the mammalian G.alpha. subunit falls at the end of
the short .alpha.C helix and therefore may distort it and
destabilize the protein.
[0226] The failure of the GPA.sub.ID and GPA.sub.LW hybrids is
predicted to be due to disruption of critical structural elements
in the i1 region as discussed above. Based upon new alignments and
the data presented in Noel et al (1993), Lambright et al (1994),
and Coleman et al (1994), this problem can be averted with the
following hybrids. In these hybrids, the junctions between the
ras-like core domain and the i1 helical domain are introduced
outside of known structural elements like alpha-helices.
[0227] Hybrid A G.alpha.S1-67/GPA66-299/G.alpha.S203-394
[0228] This hybrid contains the entire i1 insert of GPA1 interposed
into the G.alpha.S sequence.
[0229] Hybrid B
GPA1-41/G.alpha.S4443-67/GPA66-299/G.alpha.S203-394
[0230] This hybrid contains the amino terminal 41 residues of GPA1
in place of the 42 amino terminal residues of G.alpha.S found in
Hybrid A.
[0231] G.alpha.s Hybrids. There is evidence that the "switch
region" encoded by residues 171-237 of G.alpha. transducin (using
the numbering of Noel et al (1993)) also plays a role in
G.beta..gamma. coupling. First, the G226A mutation in G.alpha.S
(Miller et al. 1988) prevents the GTP-induced conformational change
that occurs with exchange of GDP for GTP upon receptor activation
by ligand. This residue maps to the highly conserved sequence
-DVGGQ-, present in all G.alpha. subunits and is involved in GTP
hydrolysis. In both the G.alpha.t and G.alpha.i1 crystal
structures, this sequence motif resides in the loop that connects
the.sub.--.beta.3 sheet and the .alpha.2 helix in the guanine
nucleotide binding core. In addition to blocking the conformational
change that occurs upon GTP binding, this mutation also prevents
dissociation of GTP-liganded G.alpha.s from G.beta..gamma.. Second,
crosslinking data reveals that a highly conserved cysteine residue
in the .alpha.2 helix (C215 in G.alpha.o, C210 in G.alpha.t) can be
crosslinked to the carboxy terminal region of G.beta._subunits.
Finally, genetic evidence (Whiteway et al. 1993) identifies an
important single residue in GPA1 (E307) in the .beta.2 sheet of the
core structure that may be in direct contact with .beta..gamma.. A
mutation in the GPA1 protein at this position suppresses the
constitutive signalling phenotype of a variety of STE4 (G.beta.)
dominant negative mutations that are also known to be defective in
G.alpha.-G.beta..gamma. association (as assessed in two-hybrid
assay in yeast as well as by more conventional genetic tests).
[0232] We have tested the hypothesis that there are switch region
determinants involved in the association of G.alpha. with
G.beta..gamma. by constructing a series of hybrid G.alpha. proteins
encoding portions of GPA1 and G.alpha.S in different combinations
(FIG. 11). The hybrid proteins were tested in both biological assay
described above and the results are summarized in Table 6.
[0233] Two conclusions may be drawn. First, in the context of the
amino terminus of G.alpha.S, the GPA1 switch region suppresses
coupling to yeast G.beta..gamma. (SGS), while in the context of the
GPA1 amino terminus the GPA1 switch region stabilizes coupling with
G.beta..gamma. (GP.sub.41-SGS). This suggests that these two
regions of GPA1 collaborate to allow interactions between G.alpha.
subunits and G.alpha..gamma. subunits. This conclusion is somewhat
mitigated by the observation that the GPA4-G.alpha.s hybrid that
does not contain the GPA1 switch region is able to complement the
growth arrest phenotype of gpa1 strains. We have not to date noted
a quantitative difference between the behaviour of the
GPA41-G.alpha.s allele and the GPA.sub.41-SGS allele, but if this
interaction is somewhat degenerate, then it may be difficult to
quantitate this accurately. The second conclusion that can be drawn
from these results is that there are other determinants involved in
stabilizing the interaction of G.alpha. with G.beta..gamma. beyond
these two regions as none of the GPA1/G.alpha.s hybrid proteins
couple as efficiently to yeast G.beta..gamma. as does native
GPA1.
[0234] The role of the surface-exposed residues of this region may
be crucial for effective coupling to yeast G.beta..gamma., and can
be incorporated into hybrid molecules as follows below.
[0235] G.alpha.S-GPA-Switch G.alpha.S 1-202/GPA298-350/G.alpha.S
253-394
[0236] This hybrid encodes the entire switch region of GPA 1 in the
context of GaS.
[0237] G.alpha.S-GPA-.alpha.2 G.alpha.S 1-226/GPA322-332/G.alpha.S
238-394
[0238] This hybrid encodes the a.sup.2 belix of GPA1 in the context
of GaS.
[0239] GPA41-G.alpha.S-GPA-.alpha.2
GPA1-41/G.alpha.S43-226/GPA322-332/G.a- lpha.S238-394
[0240] This hybrid encodes the 41 residue amino terminal domain of
GPA1 and the .alpha.2 helix of GPA1 in the context of
G.alpha.S.
[0241] Finally, the last class of hybrids that will be discussed
here are those that alter the surface exposed residues of the
.beta.2 and .beta.3 sheets of .alpha.S so that they resemble those
of the GPA1 .alpha.s helix. These altered .alpha.2 helical domains
have the following structure. (The positions of the altered
residues correspond to G.alpha.S.)
5 L203K K211E D215G K216S D229S
[0242] These single mutations can be engineered into a G.alpha.S
backbone singly and in pairwise combinations. In addition, they can
be introduced in the context of both the full length G.alpha.S and
the GPA.sub.41-G.alpha.S hybrid described previously. All are
predicted to improve the coupling of G.alpha. subunits to yeast
G.beta..gamma. subunits by virtue of improved electrostatic and
hydrophobic contacts between this region and the regions of G.beta.
defined by Whiteway and co-workers (Whiteway et al (1994) that
define site(s) that interact with GPA1).
[0243] Summary. Identification of hybrid G.alpha. subunits that
couple to the yeast pheromone pathway has led to the following
general observations. First, all GPA.sub.BamHI hybrids associate
with yeast G.beta..gamma., therefore at a minimum these hybrids
contain the determinants in GPA1 necessary for coupling to the
pheromone response pathway. Second, the amino terminal 41 residues
of GPA1 contain sufficient determinants to facilitate coupling of
G.alpha. hybrids to yeast G.beta..gamma. in some, but not all,
instances, and that some G.alpha. subunits contain regions outside
of the first 41 residues that are sufficiently similar to those in
GPA1 to facilitate interaction with GPA1 even in the absence of the
amino terminal 41 residues of GPA1. Third, there are other
determinants in the first 310 residues of GPA1 that are involved in
coupling G.alpha. subunits to yeast G.beta..gamma. subunits.
[0244] The vavious classes of hybrids noted above are not mutually
exclusive. For example, a G.alpha. containing GPA1-41 could also
feature the L203K mutation.
[0245] While, for the sake of simplicity, we have described hybrids
of yeast GPA1 and a mammalian G.alpha.s, it will be appreciated
that hybrids may be made of other yeast G.alpha. subunits and/or
other mammalian G.alpha. subunits, notably mammalian G.alpha.i
subunits. Moreover, while the described hybrids are constructed
from two parental proteins, hybrids of three or more parental
proteins are also possible.
[0246] As shown in the Examples, chimeric G.alpha. subunits have
been especially useful in coupling receptors to G.alpha.i
species.
[0247] Expression of G.alpha.
[0248] Kang et al. (1990) reported that several classes of native
mammalian G.alpha. subunits were able to interact functionally with
yeast .beta..gamma. subunits when expression of G.alpha. was driven
from a constitutively active, strong promotor (PGK) or from a
strong inducible promotor (CUP). These authors reported that rat
G.alpha.S, G.alpha.i2 or G.alpha.o expressed at high level coupled
to yeast .beta..gamma.. High level expression of mammalian
G.alpha._(i.e. non-stoichiometric with respect to yeast
.beta..gamma.) is not desirable for uses like those described in
this application. Reconstruction of G protein-coupled receptor
signal transduction in yeast requires the signalling component of
the hecerotrimeric complex (G.beta..gamma.) to be present
stoichiometrically with G.alpha. subunits. An excess of G.alpha.
subunits (as was required for coupling of mammalian G.beta.i2 and
G.alpha.o to yeast G.beta..gamma._in Kang et al.) would dampen the
signal in systems where G.beta..gamma. subunits transduce the
signal. An excess of G.beta..gamma. subunits raises the background
level of signalling in the system to unacceptably high levels.
[0249] Preferably, levels of G.alpha. and G.beta..gamma. subunits
are balanced. For example, heterologous G.alpha. subunits may be
expressed from a low copy (CEN ARS) vector containing the
endogenous yeast GPA1 promotor and the GPA1 3' untranslated region.
The minimum criterion, applied to a heterologous G.alpha. subunit
with respect to its ability to couple functionally to the yeast
pheromone pathway, is that it complement a gpa1 genotype when
expressed from the GPA1 promoter on low copy plasmids or from an
integrated, single copy gene. In the work described in this
application, all heterologous G.alpha. subunits have been assayed
in two biological systems. In the first assay heterologous G.alpha.
subunits are tested for an ability to functionally complement the
growth arrest phenotype of gpa1 strains. In the second assay the
transcription of a fus1-HIS3 reporter gene is used to measure the
extent to which the pheromone response pathway is activated, and
hence the extent to which the heterologous G.alpha. subunit
sequesters the endogenous yeast G.beta..gamma. complex.
[0250] Mammalian G.alpha.s, G.alpha.i2, G.alpha.i3, G.alpha.q,
G.alpha.11, G.alpha.16, G.alpha.o.sub.a, G.alpha.o.sub.b, and
G.alpha.z from rat, murine or human origins were expressed from a
low copy, CEN ARS vector containing the GPA1 promoter. Functional
complementation of gpa1 strains was not observed in either assay
system with any of these full-length G.alpha. constructs with the
exception of rat and human G.alpha.S.
[0251] Chimeric Yeast .beta..gamma. Subunits
[0252] An alternative to the modification of a mammalian G.alpha.
subunit for improved signal transduction is the modification of the
pertinent sites in the yeast G.beta. or G.gamma. subunits. The
principles discussed already with respect to G.alpha. subunits
apply, mutatis mutandis, to yeast G.beta. or G.gamma..
[0253] For example, it would not be unreasonable to target the
yeast Ste4p G.beta. subunit with cassette mutagenesis.
Specifically, the region of Ste4p that encodes several of the
dominant negative, signalling-defective mutations would be an
excellent target for cassette mutagenesis when looking for coupling
of yeast G.beta..gamma. to specific mammalian G.alpha.
subunits.
[0254] Protein Kinases
[0255] Mitogen-activated protein kinase (MAP kinase) and its
activator, MAP kinase kinase or MEK, are believed to be key
molecules in the transduction of intracellular signals in mammalian
cells. The activity of MAPK, a serine/threonine protein kinase, has
been shown to depend on its phosphorylation by the dually specific
MEK at tyrosine and threonine residues. MEK activity, in turn,
depends on its phosphorylation on serine and threonine by a third
kinase, MAP kinase kinase kinase, or MEKK, whose function in some
systems is fulfilled by the protooncogene product Raf1p.
[0256] An essential part of the S. cerevisiae pheromone signalling
pathway is comprised of a protein kinase cascade composed of the
products of the STE11, STE7, and FUS3/KSS1 genes (the latter pair
are distinct and functionally redundant). Functional studies have
established the dependence of FUS3p activity on tyrosine and
threonine phosphorylation by STE7p, whose activity is regulated by
its phosphorylation by STE11p. A second protein kinase cascade,
responsive to protein kinase C, has been identified in S.
cerevisiae. When this pathway is disrupted, yeast cells lose their
ability to grow in media of low osmotic strength. Although its
components have not been characterized to the same extent as that
of the mating pathway cascade, sequence analysis identifies BCK1p
as a MEKK, MKK1p/MKK2p as MEKs, and MPK1p as a MAPK.
[0257] Kinase signalling pathways appear to be conserved among
eukaryotes. Thus significant sequence homology is found between
Xenopus MAP kinase and the products of the following yeast kinase
genes: FUS3, KSS1, MPK1 (S. cerevisiae) and Spk1
(Schizo-saccharomyces pombe). In addition, mammalian MEK has been
found to be homologous to the products of STE7 (S. cerevisiae) and
Byr1 (S. pombe) [Crews et al. Science 258, 478 (1992)]. Functional
homologies between some kinases has been demonstrated through
substitution of heterologous kinase genes in yeast kinase deletion
mutants. Thus Xenopus MAP kinase will complement an mpk1.DELTA.
mutant in S. cerevisiae (however, this kinase will not substitute
for Fus3p or Kss1p function in the same organism) [Lee et al. Mol.
Cell. Biol. 13, 3067 (1993)]. Both mammalian and Xenopus MAP kinase
will substitute for Spk1 function in S. pombe [Neiman et al. Molec.
Biol. Cell 4, 107 (1993); Gotoh et al. Molec. Cell. Biol. 13, 6427
(1993)]. Rabbit MAP kinase kinase will complement a byr1 defect in
S. pombe but only when co-expressed with Raf1 kinase; the latter
thus appears to be a direct activator of MEK (Hughes et al. Nature
364, 349 (1993).
[0258] The use of the instant invention to screen for modulators of
human MEK is described in Example 9.
[0259] Cyclins
[0260] Members of another mammalian protein kinase family, one
active in progression through the cell cycle, hive been identified
by complementation of cell cycle kinase mutants in yeast. The human
homologue of p34cdc2 (S. pombe) and p34cdc28 (S. cerevisiae),
proteins which control the progression to DNA synthesis and mitosis
in yeast, was identified by complementation of a cdc2 mutation in
S. pombe [Lee and Nurse, Nature 327, 31-35 (1987)]. CDK2, a second
human p34 homologue, was identified by functional complementation
of p34cdc28 mutations in S. cerevisiae [Elledge and Spottswood,
EMBO J. 10, 2653 (1991)]. Activation of p34 depends on its
association with regulatory subunits, termed cyclins. Tight control
of cyclin expression as well as the inherent instability of these
proteins once expressed contribute to a regulated activation of p34
kinase and progression through the cell cycle.
[0261] A number of putative G1 human cyclins have been identified
through their ability to substitute for the yeast G1 cyclins, CLN1,
CLN2 and CLN3, in the pheromone signalling pathway. Thus human
cyclins C, D and E were identified by their ability to rescue cln
yeast from growth arrest [Lew et al., Cell 66, 1197 (1991)]. It has
also been demonstrated that other classes of human cyclins can
substitute functionally for the CLN proteins in yeast. The human
cyclins A, B1 and B2 (cyclins normally associated with governance
of mitosis) will also function as G1 cyclins in S. cerevisiae [Lew
et al., Cell 66, 1197 (1991)].
[0262] Certain cyclins are periodically accumulating regulators of
the p34 kinase (p34cdc2 in humans and p34cdc28 in S. cerevisiae).
The p34 kinase is functionally conserved from yeast to humans and
the activity of this threonine/serine-specific protein kinase is
required if cells are to progress through several checkpoints in
the cell cycle, including one that is termed START. START occurs
late in the G1 phase and is the switch point for cells between the
quiescent state and proliferation. The kinase is activated at
discrete points in the cell cycle through its association with
specific cyclins, regulatory subunits which are also functionally
conserved among eukaryotes. Three cyclins appear to operate in
progression through START in S. cerevisiae, CLN1, CLN2 and CLN3
(Hadwiger et al. 1989; Cross 1988; Nash 1988). The sequences of the
CLN proteins bear some homology to those of mammalian A- and B-type
cyclins; these proteins are believed to regulate S (DNA synthesis)
and M (mitotic) phases of the mammalian cell cycle.
[0263] Sequence comparisons among the cyclin proteins identified in
different species indicates that a region of high sequence
conservation is contained within an approximately 87 residue domain
that generally comprises central cyclin sequence but which is
located near the amino terminus of the yeast G1 cyclins. This
conserved domain is termed the "cyclin box". A second region of
homology shared by most of the cyclins is a C-terminal sequence
rich in proline, glutamate, serine, threonine and aspartate
residues flanked by basic amino acids that is termed a PEST motif
(Rogers et al. 1986). PEST motifs are found in unstable proteins
and are believed to signal for constitutive ubiquitin-mediated
degradation. The degradation of cyclins A and B is signalled via a
different sequence, a "mitotic destruction motif" (Glotzer et al.
1991), that is not shared by other mammalian cyclins.
[0264] Sequence comparisons made between the yeast CLN proteins and
the human A, B, C, D and E cyclins indicate the existence of
appreciable homologies (Lew et al. 1991). Across the most conserved
regions, including the cyclin box, human cyclin C bears 18%
sequence identity both to human cyclins D and E and to the yeast
CLN proteins. Human cyclins D and E appear to be more related to
human A- and B-type cyclins (33% identical) than to the yeast CLNs
(24% identical).
[0265] All human cyclins identified to date will substitute
functionally for yeast cyclins. In fact, the mammalian cyclins C,
D1 and E were identified through their ability to complement
defective CLN function in yeast (Lew et al. 1991). Mammalian A- and
B-type cyclins also substitute functionally for the CLN proteins in
yeast, therefore this ability cannot definitively mark a mammalian
cyclin as one that would operate in G1. However, the cyclins C, D1
and E have been shown to be expressed in G1 in mammalian cells and
the expression pattern of cyclin E during the cell cycle most
closely parallels the expression patterns observed for the yeast G1
cyclins (Lew et al. 1991; Koff et al. 1991).
[0266] In mammalian cells, cyclin C mRNA accumulates early in G1
while cyclin E accumulates late in that phase. D cyclin mRNA levels
are insufficient to allow tracking of expression patterns in human
cells and the role of this cyclin is therefore not clear (Lew et
al. 1991). In mouse cells, the D1 gene, CYL1, is expressed in the
G1 phase and the D1 gene appears to be regulated by
colony-stimulating factor 1 (Matsushime et al. 1991). Expression of
D1 cyclin is highly growth factor-dependent and therefore may not
be an integral part of the internal cell cycle control mechanism,
but may occur only in response to external signalling (Scherr
1993). The PRAD1 gene, found overexpressed in some parathyroid
adenomas is identical to the D1 gene (Motokura et al. 1991). D1 has
also been found to be over-expressed in a glioblastoma cell line
(Xiong et al. 1991) and is subject to deregulation by gene
amplification (Lammie et al. 1991; Keyomarsi and Pardee 1993).
Deregulation of D1 occurs by unknown mechanisms in some lymphomas,
squamous cell tumors and breast carcinomas (Bianchi et al. 1993).
This protein is involved in activating the growth of cells and
therefore, deregulated expression of this gene appears to be an
oncogenic event. Some evidence exists that the E-type cyclin may
function in the G1 to S transition in human cells: this cyclin
binds to and activates p34cdc2 protein in extracts of human
lymphoid cells in G1, the protein is associated with histone H1
kinase activity in HeLa cells (Koff et al. 1991) and cyclin E mRNA
is specifically expressed in late G1in HeLa cells (Lew et al.
1991).
[0267] It has been hypothesized chat p34 cdc2 acts at discrete
transition points in the cell cycle by phosphorylating varying
substrates. The phosphorylating activity is manifest upon
association of the kinase with cyclins which are differentially
expressed throughout the cycle of the cell. These different cyclins
may alter the substrate specificity of the kinase or may alter its
catalytic efficiency (Pines and Hunter 1990). Obvious potential
substrates for the cdc2 kinase are transcription factors that
control cell-cycle-stage-specific gene transcription.
[0268] Disruption of any one of the three CLN genes in yeast does
not appreciably affect cell growth, however, upon disruption of all
of the CLN genes, cells arrest in G1. In addition, in response to
mating pheromone, the CLN proteins are inhibited and yeast cell
growth is arrested. Two genes whose products inhibit cyclin
activity have been identified in S. cerevisiae. The products of the
FAR1 and FUS3 genes inhibit CLN2 and CLN3 function, respectively.
With pheromone signalling, the levels of Far1p and Fus3p increase,
the G1 cyclins do not accumulate, the CDC28p kinase remains
inactive, and cell growth is arrested in G1. These observations
suggest that inhibitors of the cyclin proteins, inhibitors of a
productive association between the cyclins and the kinase, or
inactivators of the kinase can foster cellular growth arrest.
[0269] By contrast, cyclins which are uninhibitable appear to
function as uncontrolled positive growth regulators. High level
expression of the CLN proteins is a lethal condition in yeast
cells. Data indicate that the loss of controlled expression of
cyclin D1 through chromosomal breakage, chromosomal translocation
or gene amplification can promote oncogenicity in mammalian cells
(Xiong et al. 1991; Lammie et al. 1991; Bianchi et al. 1993). In
addition, it appears that events that disrupt the control of cyclin
expression and control of cyclin function can result in bypass of
the G1 checkpoint and dysregulated cellular growth. The cyclin
proteins which operate in G1 to promote cellular proliferation
would be ideal targets for therapeutics aimed at control of cell
growth. Candidate surrogate proteins for substitution in the yeast
pathway for identification of such therapeutics include human
cyclins C, D and E. All three proteins are normally expressed
during the G1 phase of the mammalian cell cycle and are thus
candidate mediators of the commitment of cells to proliferate.
[0270] Examples of compounds which are known to act in G1 to
prevent entry into S phase are transforming growth factor .beta.
(TGF-.beta.) and rapamycin. Rapamycin, an immunosuppressant,
inhibits the activity of cyclin E-bound kinase. This macrolide acts
in G1to prevent the proliferation of IL-2-stimulated T lymphocytes
(Scherr 1993). TGF-.beta. has been shown to prevent progression
from G1 to S phase in mink lung epithelial cells (Howe et al.
1991). TGF-.beta. appears to interfere with activation of the
kinase, perhaps by reducing the stability of the complex which the
kinase forms with cyclin E (Koff 1993).
[0271] A strain of yeast cells bearing inactive CLN1, CLN2 and CLN3
genes and an integrated chimeric gene encoding a Gal1
promoter-driven human CLN sequence (see DL1 cells, Lew et al. Cell
66, 1197 (1991) will serve as a tester strain. The Gal1 promoter
permits high level expression when cells are grown in the presence
of galactose but this promoter is repressed when cells are grown on
glucose. Yeast cells so engineered are nonviable on glucose due to
an absence of expression of functional cyclin. These yeast,
however, proliferate on galactose-containing medium due to
expression of the human cyclin sequence. Exposure of this strain to
an inhibitor of cyclin function would render the cells incapable of
growth, even on galactose medium, i.e., the cells would growth
arrest in the presence or absence of galactose. This phenotype
could serve as an indication of the presence of an exogenously
applied cyclin inhibitor but would not be useful as a screen for
the identification of candidate inhibitors from members of a random
peptide library. Growth arrest of a subset of cells in an otherwise
growing population is useless as an indicator system. Therefore, in
order to identify random peptide inhibitors of mammalian cyclins, a
two stage screen is envisioned.
[0272] A two-hybrid system described by Fields and Song (Nature
340, 245 (1989) permits the detection of protein-protein
interactions in yeast. GAL4 protein is a potent activator of
transcription in yeast grown on galactose. The ability of GAL4 to
activate transcription depends on the presence of an N-terminal
sequence capable of binding to a specific DNA sequence (UAS.sub.G)
and a C-terminal domain containing a transcriptional activator. A
sequence encoding a protein, "A", may be fused to that encoding the
DNA binding domain of the GAL4 protein. A second hybrid protein may
be created by fusing sequence encoding the GAL4 transactivation
domain to sequence encoding a protein "B". If protein "A" and
protein "B" interact, that interaction serves to bring together the
two domains of GAL4 necessary to activate transcription of a
UAS.sub.G-containing gene. In addition to co-expressing plasmids
encoding both hybrid proteins, yeast strains appropriate for the
detection of protein-protein interactions using this two-hybrid
system would contain a GAL1-lacZ fusion to permit detection of
transcription from a UAS.sub.G sequence. These strains should also
be deleted for endogenous GAL4 and for GAL80, a negative regulator
of GAL4.
[0273] In a variation of the two-hybrid system just described, the
GAL4 DNA binding domain would be fused to a human cyclin sequence.
In addition, oligonucleotides encoding random peptides would be
ligated to sequence encoding the GAL4 transactivation domain.
Co-transformation of appropriate yeast strains with plasmids
encoding these two hybrid proteins and screening for yeast
expressing .beta.-galactosidase would permit identification of
yeast expressing a random peptide sequence capable of interacting
with a human cyclin. Identification of peptides with that
capability would be the goal of this first stage of screening. ii.
Once random peptides capable of interacting with a human cyclin of
interest had been identified, second stage screening could
commence. The second screen would permit the identification of
peptides that not only bound to human cyclin but, through that
interaction, inhibited cyclin activation of the cell
cycle-dependent kinase and, thus, cellular proliferation. Thus,
candidate peptides would be expressed, individually, in yeast
lacking CLN1, CLN2 and CLN3 but expressing a human CLN sequence, as
described above. Those peptides, expression of which does not
permit growth of the tester strain on galactose, would be presumed
cyclin inhibitors.
[0274] An advantage to this two-stage approach to the
identification of potential cyclin inhibitors is the high
probability that random peptide sequences selected in stage one
interact with human cyclin proteins. A subsequently determined
ability of that sequence to cause growth arrest of the tester yeast
on galactose would be a strong indication that the growth arrest
was due to a direct effect of the peptide on the cyclin and not on
another protein, e.g., the cell cycle dependent kinase. Though a
strong indication, such a result would not be an absolute
indication and verification of the inhibitory effect on cyclin
function could be obtained in vitro through biochemical assay.
[0275] Screening and Selection
[0276] A marker gene is a gene whose expression causes a phenotypic
change which is screenable or selectable. If the change is
selectable, the phenotypic change creates a difference in the
growth or survival rate between cells which express the marker gene
and those which do not. If the change is screenable, the phenotype
change creates a difference in some detectable characteristic of
the cells, by which the cells which express the marker may be
distinguished from those which do not. Selection is preferable to
screening.
[0277] The marker gene may be coupled to the yeast pheromone
pathway so that expression of the marker gene is dependent on
activation of the G protein. This coupling may be achieved by
operably linking the marker gene to a pheromone-responsive
promoter. The term "pheromone-responsive promoter" indicates a
promoter which is regulated by some product of the yeast pheromone
signal transduction pathway, not necessarily pheromone per se. In
one embodiment, the promoter is activated by the pheromone pathway,
in which case, for selection, the expression of the marker gene
should result in a benefit to the cell. A preferred marker gene is
the imidazoleglycerol phosphate dehydratase gene (HIS3). If a
pheromone responsive promoter is operably linked to a beneficial
gene, the cells will be useful in screening or selecting for
agonists. If it is linked to a deleterious gene, the cells will be
useful in screening or selecting for antagonists.
[0278] Alternatively, the promoter may be one which is repressed by
the pheromone pathway, thereby preventing expression of a product
which is deleterious to the cell. With a pheromone-repressed
promoter, one screens for agonists by linking the promoter to a
deleterious gene, and for antagonists, by linking it to a
beneficial gene.
[0279] Repression may be achieved by operably linking a
pheromone-induced promoter to a gene encoding mRNA which is
antisense to at least a portion of the mRNA encoded by the marker
gene (whether in the coding or flanking regions), so as to inhibit
translation of that mRNA. Repression may also be obtained by
linking a pheromone-induced promoter to a gene encoding a
DNA-binding repressor protein, and incorporating a suitable
operator site into the promoter or other suitable region of the
marker gene.
[0280] Suitable positively selectable (beneficial) genes include
the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1, 2, 3, 4, 5, 7,
8; ARG1, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2, 5; THR1, 4; TRP2, 3,
4, 5; LEU1, 4; MET2, 3, 4, 8, 9, 14, 16, 19; URA1, 2, 4, 5, 10;
HOM3, 6; ASP3; CHO1; ARO 2, 7; CYS3; OLE1; INO1, 2, 4; PRO1, 3
Countless other genes are potential selective markers. The above
are involved in well-characterized biosynthetic pathways.
[0281] The imidazoleglycerol phosphate dehydratase (IGP
dehydratase) gene (HIS3) is preferred because it is both quite
sensitive and can be selected over a broad range of expression
levels. In the simplest case, the cell is auxotrophic for histidine
(requires histidine for growth) in the absence of activation.
Activation leads to synthesis of the enzyme and the cell becomes
prototrophic for histidine (does not require histidine). Thus the
selection is for growth in the absence of histidine. Since only a
few molecules per cell of IGP dehydratase are required for
histidine prototrophy, the assay is very sensitive.
[0282] In a more complex version of the assay, cells can be
selected for resistance to aminotriazole (AT), a drug that inhibits
the activity of IGP dehydratase. Cells with low, fixed level of
expression of HIS3 are sensitive to the drug, while cells with
higher levels are resistant. The amount of AT can be selected to
inhibit cells with a basal level of HIS3 expression (whatever that
level is) but allow growth of cells with an induced level of
expression. In this case selection is for growth in the absence of
histidine and in the presence of a suitable level of AT.
[0283] In appropriate assays, so-called counterselectable or
negatively selectable genes may be used. Suitable genes include:
URA3 (orotidine-5'-phosphate decarboxylase; inhibits growth on
5-fluoroorotic acid), LYS2 (2-aminoadipate reductase; inhibits
growth on .alpha.-aminoadipate as sole nitrogen source), CYH2
(encodes ribosomal protein L29; cycloheximide-sensitive allele is
dominant to resistant allele), CAN1 (encodes arginine permease;
null allele confers resistance to the arginine analog canavanine),
and other recessive drug-resistant markers.
[0284] The natural response to induction of the yeast pheromone
response pathway is for cells to undergo growth arrest. This is the
preferred way to select for antagonists to a ligand/receptor pair
that induces the pathway. An autocrine peptide antagonist would
inhibit the activation of the pathway; hence, the cell would be
able to grow. Thus, the FAR1 gene may be considered an endogenous
counterselectable marker. The FAR1 gene is preferably inactivated
when screening for agonist activity.
[0285] The marker gene may also be a screenable gene. The screened
characteristic may be a change in cell morphology, metabolism or
other screenable features. Suitable markers include
beta-galactosidase (Xgal, C.sub.12FDG, Salmon-gal, Magenta-Gal
(latter two from Biosynth Ag)), alkaline phosphatase, horseradish
peroxidase, exo-glucanase (product of yeast exb1 gene;
nonessential, secreted); luciferase; and chloramphenicol
transferase. Some of the above can be engineered so that they are
secreted (although not .beta.-galactosidase). The preferred
screenable marker gene is beta-galactosidase; yeast cells
expressing the enzyme convert the colorless substrate Xgal into a
blue pigment. Again, the promoter may be pheromone-induced or
pheromone-inhibited.
[0286] Yeast Cells
[0287] The yeast may be of any species that possess a G
protein-mediated signal transduction pathway and which are
cultivatable. Suitable species include Kluyverei lactis,
Schizosaccharomyces pombe, and Ustilago maydis; Saccharomyces
cerevisiae is preferred. Either G.alpha. or G.beta..gamma. may be
the activator of the "effecter." (It is suspected that in some
species, both G.alpha.-activated and G.beta..gamma.-activated
effectors exist.) The term "yeast", as used herein, includes not
only yeast in a strictly taxonomic sense (i.e., unicellular
organisms), but also yeast-like multicellular fungi with pheromone
responses mediated by the mating pathway.
[0288] The yeast cells of the present invention may be used to test
peptides for the ability to interact with an exogenous G
protein-coupled receptor or other PSP surrogate. The yeast cells
must express both the exogenous G protein-coupled receptor (or
other PSP surrogate), and a complementary G protein (or other PSPs
necessary for the PSP surrogate to function in the pheromone
system, if need be after activation by a drug), and these molecules
must be presented in such a manner that a "readout" can be obtained
by means of the pheromone response pathway (which may be modified
to improve the readout).
[0289] For a readout to be possible, a gene encoding a selectable
or screenable trait must be coupled to the G protein-mediated
signal transduction pathway so that the level of expression of the
gene is sensitive to the presence or absence of a signal, i.e.,
binding to the coupled exogenous receptor. This gene may be an
unmodified gene already in the pathway, such as the genes
responsible for growth arrest. It may be a yeast gene, not normally
a part of the pathway, that has been operably linked to a
"pheromone-responsive" promoter. Or it may be a heterologous gene
that has been so linked. Suitable genes and promoters were
discussed above.
[0290] It will be understood that to achieve selection or
screening, the yeast must have an appropriate phenotype. For
example, introducing a pheromone-responsive chimeric HIS3 gene into
a yeast that has a wild-type HIS3 gene would frustrate genetic
selection. Thus, to achieve nutritional selection, an auxotrophic
strain is wanted.
[0291] The yeast cells of the present invention optionally possess
one or more of the following characteristics:
[0292] (a) the endogenous FAR1 gene has been inactivated;
[0293] (b) the endogenous SST2 gene, and/or other genes involved in
desensitization, has been inactivated;
[0294] (c) the endogenous pheromone (a- or .alpha.-factor) receptor
gene has been inactivated; and
[0295] (d) the endogenous pheromone genes have been
inactivated.
[0296] "Inactivation" means that production of a functional gene
product is prevented or inhibited. Inactivation may be achieved by
deletion of the gene, mutation of the promoter so that expression
does not occur, or mutation of the coding sequence so that the gene
product is inactive. Inactivation may be partial or total.
[0297] Mutants with inactivated supersensitivity-related genes can
be identified by conventional genetic screening procedures. The
far1 gene was identified as an .alpha.-factor resistant mutant that
remained blue (with fus1-lacZ) on .alpha.-factor/Xgal. far2, as it
turns out, is the same as fus3. Supersensitive mutants could be
identified as constitutive weak blue colonies expressing fus1-lacZ
on Xgal, or as strains that can mate more proficiently with a poor
pheromone-secreter.
[0298] The DNA sequences of (a) the .alpha.- and a-factor genes,
(b) the .alpha.- and a-factor receptors, (c) the FAR1 gene, (d) the
SST2 gene, and (e) the FUS1 promoter have been reported in the
following references:
[0299] MFa1 and MFa2: A J Brake, C Brenner, R Najarian, P Laybourn,
and J Merryweather. Structure of Genes Encoding Precursors of the
Yeast Peptide Mating Pheromone a-Factor. In Protein Transport and
Secretion. Gething M-J, ed. Cold Spring Harbor Lab, New York,
1985.
[0300] MF.alpha.1 and MF.alpha.2: Singh, A. E Y Chen, J M Lugovoy,
C N Chang, R A Hitzeman et al. 1983. Saccharomyces cerevisiae
contains two discrete genes coding for the .alpha.-pheromone.
Nucleic Acids Res. 11:4049; J Kurjan and I Herskowitz. 1982.
Structure of a yeast pheromone gene (MF): A putative .alpha.-factor
precursor contains four tandem copies of mature .alpha.-factor.
Cell 30:933.
[0301] STE2 and STE3: A C Burkholder and L H Hartwell. 1985. The
yeast .alpha.-factor receptor: Structural properties deduced from
the sequence of the STE2 gene. Nucleic Acids Res. 13:8463; N
Nakayama, A Miyajima, and K Arai. 1985. Nucleotide sequences of
STE2 and STE3, cell type-specific sterile genes from Saccharomyces
cerevisiae. EMBO J. 4:2643; D C Hagen, G McCaffrey, and G F
Sprague, Jr. 1986. Evidence the yeast STE3 gene encodes a receptor
for the peptide pheromone a-factor: Gene sequence and implications
for the structure of the presumed receptor. Proc Natl Acad Sci
83:1418.
[0302] FAR1: F Chang and I Herskowitz. 1990. Identification of a
gene necessary for cell cycle arrest by a negative growth factor of
yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell 63:999.
[0303] SST2: C Dietzel and J Kurjan. 1987. Pheromonal regulation
and sequence of the Saccharomyces cerevisiae SST2 gene: A model for
desensitization to pheromone. Mol Cell Biol 7: 4169.
[0304] FUS1: J Trueheart, J D Boeke, and G R Fink. 1987. Two genes
required for cell fusion during yeast conjugation: Evidence for a
pheromone-induced surface protein. Mol Cell Biol 7:2316.
[0305] The various essential and optional features may be imparted
to yeast cells by, e.g., one or more of the following means:
isolation of spontaneous mutants with one or more of the desired
features; mutation of yeast by chemical or radiation treatment,
followed by selection; and genetic engineering of yeast cells to
introduce, modify or delete genes.
[0306] Other explicit characteristics desirable in strains of yeast
designed to be used as screening devices for inhibitors/activators
of PSP surrogates are discussed in subsections dealing specifically
with each molecular target.
[0307] Peptide
[0308] The term "peptide" is used herein to refer to a chain of two
or more amino acids, with adjacent amino acids joined by peptide
(--NHCO--) bonds. Thus, the peptides of the present invention
include oligopeptides, polypeptides, and proteins. Preferably, the
peptides of the present invention are 2 to 200, more preferably 5
to 50, amino acids in length. The minimum peptide length is chiefly
dictated by the need to obtain sufficient potency as an activator
or inhibitor. The maximum peptide length is only a function of
synthetic convenience once an active peptide is identified.
[0309] For initial studies in which the cognate PSP was a yeast
pheromone receptor, a 13-amino acid peptide was especially
preferred as that is the length of the mature yeast
.alpha.-factor.
[0310] Peptide Libraries
[0311] A "peptide library" is a collection of peptides of many
different sequences (typically more than 1000 different sequences),
which are prepared essentially simultaneously, in such a way that,
if tested simultaneously for some activity, it is possible to
characterize the "positive" peptides.
[0312] The peptide library of the present invention takes the form
of a yeast cell culture, in which essentially each cell expresses
one, and usually only one, peptide of the library. While the
diversity of the library is maximized if each cell produces a
peptide of a different sequence, it is usually prudent to construct
the library so there is some redundancy.
[0313] In the present invention, the peptides of the library are
encoded by a mixture of DNA molecules of different sequence Each
peptide-encoding DNA molecule is ligated with a vector DNA molecule
and the resulting recombinant DNA molecule is introduced into a
yeast cell. Since it is a matter of chance which peptide-encoding
DNA molecule is introduced into a particular cell, it is not
predictable which peptide that cell will produce. However, based on
a knowledge of the manner in which the mixture was prepared, one
may make certain statistical predictions about the mixture of
peptides in the peptide library.
[0314] It is convenient to speak of the peptides of the library as
being composed of constant and variable residues. If the nth
residue is the same for all peptides of the library, it is said to
be constant. If the nth residue varies, depending on the peptide in
question, the residue is a variable one. The peptides of the
library will have at least one, and usually more than one, variable
residue. A variable residue may vary among any of two to all twenty
of the genetically encoded amino acids; the variable residues of
the peptide may vary in the same or different manner. Moreover, the
frequency of occurrence of the allowed amino acids at a particular
residue position may be the same or different. The peptide may also
have one or more constant residues.
[0315] There are two principal ways in which to prepare the
required DNA mixture. In one method, the DNAs are synthesized a
base at a time. When variation is desired, at a base position
dictated by the Genetic Code, a suitable mixture of nucleotides is
reacted with the nascent DNA, rather than the pure nucleotide
reagent of conventional polynucleotide synthesis.
[0316] The second method provides more exact control over the amino
acid variation. First, trinucleotide reagents are prepared, each
trinucleotide being a codon of one (and only one) of the amino
acids to be featured in the peptide library. When a particular
variable residue is to be synthesized, a mixture is made of the
appropriate trinucleotides and reacted with the nascent DNA.
[0317] Once the necessary "degenerate" DNA is complete, it must be
joined with the DNA sequences necessary to assure the expression of
the peptide, as discussed in more detail below, and the complete
DNA construct must be introduced into the yeast cell.
[0318] Expression System
[0319] The expression of a peptide-encoding gene in a yeast cell
requires a promoter which is functional in yeast. Suitable
promoters include the promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255,
2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.
Enzyme Reg. 7, 149 (1968); and Holland et al. Biochemistry 17, 4900
(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Suitable vectors and promoters for use in yeast
expression are further described in R. Hitzeman et al., EPO Publn.
No. 73,657. Other promoters, which have the additional advantage of
transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned metallothionein and
glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes
responsible for maltose and galactose utilization. Finally,
promoters that are active in only one of the two haploid mating
types may be appropriate in certain circumstances. Among these
haploid-specific promoters, the pheromone promoters MFa1 and
MF.alpha.1 are of particular interest.
[0320] In screens devised for a subset of PSP surrogates (e.g.
kinases, cyclins) random peptide sequences need not be expressed in
the context of yeast pheromone and need not be engineered for
secretion or transport to the extracellular space. Libraries of
random peptides may be expressed in a multiplicity of ways,
including as portions of chimeric proteins, as in a two-hybrid
protein system designed to signal protein-protein interactions.
Random peptides need not necessarily substitute for yeast
pheromones but can impinge on the pheromone pathway downstream of
the interaction between pheromone and pheromone receptor (as in
random peptide inhibitors of the kinases or of the cyclins).
[0321] In constructing suitable expression plasmids, the
termination sequences associated with these genes, or with other
genes which are efficiently expressed in yeast, may also be ligated
into the expression vector 3' of the heterologous coding sequences
to provide polyadenylation and termination of the mRNA.
[0322] Vectors
[0323] The vector must be capable of replication in a yeast cell.
It may be a DNA which is integrated into the host genome, and
thereafter is replicated as a part of the chromosomal DNA, or it
may be DNA which replicates autonomously, as in the case of a
plasmid. In the latter case, the vector must include an origin of
replication which is functional in the host. In the case of an
integrating vector, the vector may include sequences which
facilitate integration, e.g., sequences homologous to host
sequences, or encoding integrases.
[0324] Besides being capable of replication in yeast cells, it is
convenient if the vector can also be replicated in bacterial cells,
as many genetic manipulations are more conveniently carried out
therein. Shuttle vectors capable of replication in both yeast and
bacterial cells include YEps, YIps, and the pRS series.
[0325] Periplasmic Secretion
[0326] The cytoplasm of the yeast cell is bounded by a lipid
bilayer called the plasma membrane. Between this plasma membrane
and the cell wall is the periplasmic space. Peptides secreted by
yeast cells cross the plasma membrane through a variety of
mechanisms and thereby enter the periplasmic space. The secreted
peptides are then free to interact with other molecules that are
present in the periplasm or displayed on the outer surface of the
plasma membrane. The peptides then either undergo re-uptake into
the cell, diffuse through the cell wall into the medium, or become
degraded within the periplasmic space.
[0327] The peptide library may be secreted into the periplasm by
one of two distinct mechanisms, depending on the nature of the
expression system to which they are linked. In one system, the
peptide may be structurally linked to a yeast signal sequence, such
as that present in the .alpha.-factor precursor, which directs
secretion through the endoplasmic reticulum and Golgi apparatus.
Since this is the same route that the receptor protein follows in
its journey to the plasma membrane, opportunity exists in cells
expressing both the receptor and the peptide library for a specific
peptide to interact with the receptor during transit through the
secretory pathway. This has been postulated to occur in mammalian
cells exhibiting autocrine activation. Such interaction would
likely yield activation of the linked pheromone response pathway
during transit, which would still allow identification of those
cells expressing a peptide agonist. For situations in which peptide
antagonists to externally applied receptor agonist are sought, this
system would still be effective, since both the peptide antagonist
and receptor would be delivered to the outside of the cell in
concert. Thus, those cells producing an antagonist would be
selectable, since the peptide antagonist would be properly and
timely situated to prevent the receptor from being stimulated by
the externally applied agonist.
[0328] An alternative mechanism for delivering peptides to the
periplasmic space is to use the ATP-dependent transporters of the
STE6/MDR1 class. This transport pathway and the signals that direct
a protein or peptide to this pathway are not as well characterized
as is the endoplasmic reticulum-based secretory pathway.
Nonetheless, these transporters apparently can efficiently export
certain peptides directly across the plasma membrane, without the
peptides having to transit the ER/Golgi pathway. We anticipate that
at least a subset of peptides can be secreted through this pathway
by expressing the library in context of the a-factor prosequence
and terminal tetrapeptide. The possible advantage of this system is
that the receptor and peptide do not come into contact until both
are delivered to the external surface of the cell. Thus, this
system strictly mimics the situation of an agonist or antagonist
that is normally delivered from outside the cell. Use of either of
the described pathways is within the scope of the invention.
[0329] The present invention does not require periplasmic
secretion, or, if such secretion is provided, any particular
secretion signal or transport pathway.
EXAMPLE 1
Development of Autocrine Yeast Strains
[0330] In this example, we describe a pilot experiment in which
haploid cells were engineered to be responsive to their own
pheromones (see FIG. 1). (Note that in the examples, functional
genes are capitalized and inactivated genes are in lower case.) For
this purpose we constructed recombinant DNA molecules designed
to:
[0331] i. place the coding region of STE2 under the transcriptional
control of elements which normally direct the transcription of
STE3. This is done in a plasmid that allows the replacement of
genomic STE3 of S. cerevisiae with sequences wherein the coding
sequence of STE2 is driven by STE3 transcriptional control
elements.
[0332] ii. place the coding region of STE3 under the
transcriptional control of elements which normally direct the
transcription of STE2. This is done in a plasmid which will allow
the replacement of genomic STE2 of S. cerevisiae with sequences
wherein the coding sequence of STE3 is driven by STE2
transcriptional control elements.
[0333] The sequence of the STE2 gene is known see Burkholder A. C.
and Hartwell L. H. (1985), "The yeast .alpha.-factor receptor:
Structural properties deduced from the sequence of the STE2 gene,"
Nuc. Acids Res. 13, 8463; Nakayama N., Miyajima A., Arai K. (1985)
"Nucleotide sequences of STE2 and STE3, cell type-specific sterile
genes from Saccharomyces cerevisiae," EMBO J. 4, 2643.
[0334] A 4.3 kb BamHI fragment that contains the entire STE2 gene
was excised from plasmid YEp24-STE2 (obtained from J. Thorner,
Univ. of California) and cloned into PALTER (Protocols and
Applications Guide, 1991, Promega Corporation, Madison, Wis.). An
SpeI site was introduced 7 nucleotides (nts) upstream of the ATG of
STE2 with the following mutagenic oligonucleotide, using the STE2
minus strand as template (mutated bases are underlined and the
start codon is in bold type):
5'GTTAAGAACCATATACTAGTATCAAAAATGTCTG3' (SEQ ID NO:14).
[0335] A second SpeI site was simultaneously introduced just
downstream of the STE2 stop codon with the following mutagenic
oligonucleotide (mutated bases are underlined and the stop codon is
in bold type): 5'TGATCAAAATTTACTAGTTTGAAAAAGTAATTTCG3' (SEQ ID
NO:15).
[0336] The BamHI fragment of the resulting plasmid (Cadus 1096),
containing STE2 with SpeI sites immediately flanking the coding
region, was then subcloned into the yeast integrating vector YIp19
to yield Cadus 1143.
[0337] The STE3 sequence is also known. Nakayama N., Miyajima A.,
Arai K. (1985), "Nucleotide sequences of STE2 and STE3, cell
type-specific sterile genes from Saccharomyces cerevisiae," EMBO J.
4, 2643; Hagen D. C., McCaffrey G., Sprague G. F. (1986), "Evidence
the yeast STE3 gene encodes a receptor for the peptide pheromone
a-factor: gene sequence and implications for the structure of the
presumed receptor," Proc. Natl. Acad. Sci. 83, 1418. STE3 was made
available by Dr. J. Broach as a 3.1 kb fragment cloned into
pBLUESCRIPT-KS II (Stratagene, 11011 North Torrey Pines Road, La
Jolla, Calif. 92037). STE3 was subcloned as a KpnI-XbaI fragment
into both M13mp18 RF (to yield Cadus 1105) and pUC19 (to yield
Cadus 1107). The two SpeI sites in Cadus 1107 were removed by
digestion with SpeI, fill-in with DNA polymerase I Klenow fragment,
and recircularization by blunt-end ligation. Single-stranded DNA
containing the minus strand of STE 3 was obtained using Cadus 1105
and SpeI sites were introduced 9 nts upstream of the start codon
and 3 nts downstream of the stop codon of STE3 with the following
mutagenic oligonucleotides, respectively:
6 5' GGCAAAATACTAGTAAAATTTTCATGTC 3' (SEQ ID NO:16). 5'
GGCCCTTAACACACTAGTGTCGCATTATATTTAC 3' (SEQ ID NO:17).
[0338] The mutagenesis was accomplished using the T7-GEN protocol
of United States Biochemical (T7-GEN In Vitro Mutagenesis Kit,
Descriptions and Protocols, 1991, United States Biochemical, P.O.
Box 22400, Cleveland, Ohio 44122). The replicative form of the
resulting Cadus 1141 was digested with AflII and KpnI, and the
approximately 2 kb fragment containing the entire coding region of
STE3 flanked by the two newly introduced Spe I sites was isolated
and ligated with the approximately 3.7 kb vector fragment of AflII-
and KpnI-digested Cadus 1107, to yield Cadus 1138. Cadus 1138 was
then digested with XbaI and KpnI, and the STE3-containing 2.8 kb
fragment was ligated into the XbaI- and KpnI-digested yeast
integrating plasmid pRS406 (Sikorski, R. S. and Hieter, P. (1989)
"A System of Shuttle Vectors and Yeast Host Strains Designed for
Efficient Manipulation of DNA in Saccharomyces cerevisiae",
Genetics 122:19-27 to yield Cadus 1145.
[0339] The SpeI fragment of Cadus 1143 was replaced with the SpeI
fragment of Cadus 1145 to yield Cadus 1147, in which the coding
sequences of STE3 are under the control of STE2 expression
elements. Similarly, the SpeI fragment of Cadus 1145 was replaced
with the SpeI fragment of Cadus 1143 to yield Cadus 1148, in which
the coding sequences of STE2 are under the control of STE3
expression elements. Using the method of pop-in/pop-out replacement
(Rothstein, R. (1991) "[19] Targeting, Disruption, Replacement, and
Allele Rescue: Integrative DNA Transformation in Yeast", Methods in
Enzymology, 194:281-301), Cadus 1147 was used to replace genomic
STE2 with the ste2-STE3 hybrid in a MATa cell and Cadus 1148 was
used to replace genomic STE3 with the ste3-STE2 hybrid in a
MAT.alpha. cell. Cadus 1147 and 1148 contain the selectable marker
URA3.
[0340] Haploid yeast of mating type a which had been engineered to
express HIS3 under the control of the pheromone-inducible FUS1
promoter were transformed with CADUS 1147, and transformants
expressing URA3 were selected. These transformants, which express
both Ste2p and Ste3p, were plated on 5-fluoroorotic acid to allow
the selection of clones which had lost the endogenous STE2, leaving
in its place the heterologous, integrated STE3. Such cells
exhibited the ability to grow on media deficient in histidine,
indicating autocrine stimulation of the pheromone response
pathway.
[0341] Similarly, haploids of mating type .alpha. that can express
HIS3 under the control of the pheromone-inducible FUS1 promoter
were transformed with CADUS 1148 and selected for replacement of
their endogenous STE3 with the integrated STE2. Such cells showed,
by their ability to grow on histidine-deficient media, autocrine
stimulation of the pheromone response pathway.
EXAMPLE 2
Strain Development
[0342] In this example, yeast strains are constructed which will
facilitate selection of clones which exhibit autocrine activation
of the pheromone response pathway. To construct appropriate yeast
strains, we will use: the YIp-STE3 and pRS-STE2 knockout plasmids
described above, plasmids available for the knockout of FAR1, SST2,
and HIS3, and mutant strains that are commonly available in the
research community. The following haploid strains will be
constructed, using one-step or two-step knockout protocols
described in Meth. Enzymol 194:281-301, 1991:
7 1. MAT.alpha. ste3::STE2::ste3 far1 sst2 FUS1::HIS3 2. MATa
ste2::STE3::ste2 far1 sst2 FUS1::HIS3 3. MAT.alpha.
ste3::STE2::ste3 far1 sst2 mf.alpha.1 mf.alpha.2 FUS1::HIS3 4. MATa
ste2::STE3::ste2 far1 sst2 mfa1 mfa2 FUS1::HIS3 5. MATa bar1 far1-1
fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3 6. MATa mfa1 mfa2 far1-1
his3::fus1-HIS3 ste2-STE3 ura3 met1 ade1 leu2
[0343] Strains 1 and 2 will be tested for their ability to grow on
histidine-deficient media as a result of autocrine stimulation of
their pheromone response pathways by the pheromones which they
secrete. If these tests prove successful, strain 1 will be modified
to inactivate endogenous MF.alpha.1 and MF.alpha.2. The resulting
strain 3, MAT.alpha. far1 sst2 ste3::STE2::ste3 FUS1::HIS3 mfa1
mfa2, should no longer display the selectable phenotype (i.e., the
strain should be auxotrophic for histidine). Similarly, strain 2
will be modified to inactivate endogenous MFa1 and MFa2. The
resulting strain 4, MATa far1 sst2 ste2::STE3::ste2 FUS1::HIS3 mfa1
mfa2, should be auxotrophic for histidine. The uses of strains 5
and 6 are outlined in Examples 3 and 4 below.
EXAMPLE 3
Peptide Library
[0344] In this example, a synthetic oligonucleotide encoding a
peptide is expressed so that the peptide is secreted or transported
into the periplasm.
[0345] i. The region of MF.alpha.1 which encodes mature
.alpha.-factor has been replaced via single-stranded mutagenesis
with restriction sites that can accept oligonucleotides with AflII
and BglII ends. Insertion of oligonucleotides with AflII and BglII
ends will yield plasmids which encode proteins containing the
MF.alpha.1 signal and leader sequences upstream of the sequence
encoded by the oligonucleotides. The MF.alpha.1 signal and leader
sequences should direct the processing of these precursor proteins
through the pathway normally used for the transport of mature
.alpha.-factor.
[0346] The MF.alpha.1 gene, obtained as a 1.8 kb EcoRI fragment
from pDA6300 (J. Thorner, Univ. of California) was cloned into
pALTER (see FIG. 2) in preparation for oligonucleotide-directed
mutagenesis to remove the coding region of mature .alpha.-factor
while constructing sites for acceptance of oligonucleotides with
AflII and BclI ends. The mutagenesis was accomplished using the
minus strand as template and the following mutagenic
oligonucleotide:
8 5'CTAAAGAAGA AGGGGTATCT TTGCTTAAGC TCGAGATCTC GACTGATAAC
AACAGTGTAG 3' (SEQ ID NO:18).
[0347] A HindIII site was simultaneously introduced 7 nts upstream
of the MF.alpha.1 start codon with the oligonucleotide:
5'CATACACAAT ATAAAGCTTT AAAAGAATGA G3' (SEQ ID NO:19).
[0348] The resulting plasmid, Cadus 1214, contains a HindIII site 7
nts upstream of the MF.alpha.1 initiation codon, an AflII site at
the positions which encode the KEX2 processing site in the
MF.alpha.1 leader peptide, and XhoI and BglII sites in place of all
sequences from the leader-encoding sequences up to and including
the normal stop codon. The 1.5 kb HindIII fragment of Cadus 1214
therefore provides a cloning site for oligonucleotides to be
expressed in yeast and secreted through the pathway normally
travelled by endogenous .alpha.-factor.
[0349] A sequence comprising the ADH1 promoter and 5' flanking
sequence was obtained as a 1.5 kb BamHI-HindIII fragment from pAAH5
(Ammerer, G. (1983) "[11] Expression of Genes in Yeast Using the
ADCI Promoter", Academic Press, Inc., Meth. Enzymol. 101, 192-201
and ligated into the high copy yeast plasmid pRS426 (Christianson,
T. W et al. (1992) "Multifunctional yeast high-copy-number shuttle
vectors", Gene 110:119-122) (see FIG. 3). The unique XhoI site in
the resulting plasmid was eliminated to yield Cadus 1186. The 1.5
Kb HindIII fragment of Cadus 1214 was inserted into
HindIII-digested Cadus 1186; expression of sequences cloned into
this cassette initiates from the ADH1 promoter. The resulting
plasmid, designated Cadus 1215, can be prepared to accept
oligonucleotides with AflII and BclI ends by digestion with those
restriction endonucleases. The oligonucleotides will be expressed
in the context of MF.alpha.1 signal and leader peptides (FIG.
4).
[0350] Modified versions of Cadus 1215 were also constructed. To
improve the efficiency of ligation of oligonucleotides into the
expression vector, Cadus 1215 was restricted with KpnI and
religated to yield Cadus 1337. This resulted in removal of one of
two HindIII sites. Cadus 1337 was linearized with HindIII,
filled-in, and recircularized to generate Cadus 1338. To further
tailor the vector for library construction, the following
double-stranded oligonucleotide was cloned into AflII- and
BglII-digested Cadus 1338:
9 5' TTAAGCGTGAGGCAGAAGCTTATCGATA oligo 062 (SEQ ID NO:37) 3'
CGCACTCCGTCTTCGAATAGCTATCTAG oligo 063 (SEQ ID NO:38)
[0351] The HindIII site is italicized and a ClaI site is
emboldened; this ClaI site is unique in the resulting vector, Cadus
1373. In Cadus 1373, the HindIII site that exists at the junciton
between the MF.alpha. pro sequence and the mature peptide to be
expressed by this vector was made unique. Therefore the HindIII
site and the downstream BglII site can be used to insert
oligo-nucleotides encoding peptides of interest. These
modifications of Cadus 1215 provide an laternative to the use of
the AflII site in the cloning of oligonucleotides into the
expressions vector.
[0352] Cadus 1373 was altered further to permit elimination from
restricted vector preparations of contaminating singly-cut plasmid.
Such contamination could result in unacceptably high background
transformation. To eliminate this possibility, approximately 1.1 kb
of dispensable ADH1 sequence at the 5' side of the promoter region
was deleted. This was accomplished by restruction of Cadus 1373
with SphI and BamHI, fill-in, and ligation; this maneuver
regenerates the BamHI site. The resulting vector, Cadus 1624, was
then restricted with HindIII and ClaI and an approximately 1.4 kb
HindIII and ClaI fragment encoding lacZ was inserted to generate
Cadus 1625. Use of HindIII- and BglII-restricted Cadus 1625 for
acceptance of oligonucleotides results in a low background upon
transformation of the ligation product into bacteria.
[0353] Two single-stranded oligonucleotide sequences (see below)
are synthesized, annealed, and repetitively filled in, denatured,
and reannealed to form double-stranded oligonucleotides that, when
digested with AflII and BclI, can be ligated into the polylinker of
the expression vector, Cadus 1215. The two single-stranded
oligonucleotides have the following sequences: 5'G CTA CTT AAG CGT
GAG GCA GAA GCT3' (SEQ ID NO:20) and 5'C GGA TGA TCA (NNN).sub.n
AGC TTC TGC CTC ACG CTT AAG TAG C3' (SEQ ID NO:21) where N is any
chosen nucleotide and n is any chosen integer. Yeast transformed
with the resulting plasmids will secrete--through the
.alpha.-factor secretory pathway--peptides whose amino acid
sequence is determined by the particular choice of N and n (FIG.
4).
[0354] Alternatively, the following single stranded
oligonucleotides are used:
[0355] MF.alpha.NNK (76 mer):
10 MF.alpha.NNK (76 mer): 5'CTGGATGCGAAGATCAGCTNNKNNKNNKNNK-
NNKNNKNNKNNKNNKNNKNNKNNK (SEQ ID NO:39) and TGATCAGTCTGTGACGC 3'
MF.alpha.Mbo (17 mer): 5' GCGTCACAGACTGATCA 3' (SEQ ID NO:40)
[0356] When annealed the double stranded region is:
11 TGATCAGTCTGTGACGC (last 17 bases of SEQ ID NO:39)
ACTAGTCAGACACTGCG (SEQ ID NO:40 in 3' to 5' direction)
[0357] where the FokI site is underlined, the BbsI site is
emboldened, and the MboI site is italicized. After fill-in using
Taq DNA polymerase (Promega Corporation, Madison, Wis.), the double
stranded product is restricted with BbsI and MboI and ligated to
HindIII- and BglII-restricted Cadus 1373.
[0358] ii. The region of MFa1 which encodes mature a-factor will be
replaced via single stranded mutagenesis with restriction sites
that can accept oligonucleotides with XhoI and AflII ends.
[0359] Insertion of oligonucleotides with XhoI and AflII ends will
yield plasmids which encode proteins containing the MFa1 leader
sequences upstream of the sequence encoded by the oligonucleotides.
The MFa1 leader sequences should direct the processing of these
precursor proteins through the pathway normally used for the
transport of mature a-factor. MFA1, obtained as a BamHI fragment
from pKK1 (provided by J. Thorner and K. Kuchler), was ligated into
the BamHI site of pALTER (Promega) (FIG. 5). Using the minus strand
of MFA1 as template, a HindIII site was inserted by
oligonucleotide-directed mutagenesis just 5' to the MFA1 start
codon using the following oligonucleotide:
5'CCAAAATAAGTACAAAGCTTTC- GAATAGAAATGCAACCATC (SEQ ID NO:22). A
second oligonucleotide was used simultaneously to introduce a short
polylinker for later cloning of synthetic oligonucleotides in place
of MFA1 sequences. These MFA1 sequences encode the C-terminal 5
amino acids of the 21 amino acid leader peptide through to the stop
codon: 5'GCCGCTCCAAAAGAAAAGACCTCGAGCTCGCTTAAG- TTCTGCGTACA
AAAACGTTGTTC3' (SEQ ID NO:23). The 1.6 kb HindIII fragment of the
resulting plasmid, Cadus 1172, contains sequences encoding the MFA1
start codon and the N-terminal 16 amino acids of the leader
peptide, followed by a short polylinker containing XhoI, SacI, and
AflII sites for insertion of oligonucleotides. The 1.6 kb HindIII
fragment of Cadus 1172 was ligated into HindIII-digested Cadus 1186
(see above) to place expression of sequences cloned into this
cassette under the control of the ADH1 promoter. The SacI site in
the polylinker was made unique by eliminating a second SacI site
present in the vector. The resulting plasmid, designated Cadus
1239, can be prepared to accept oligonucleotides with XhoI and
AflII ends by digestion with those restriction endonucleases for
expression in the context of MFa1 leader peptides (FIG. 6).
[0360] Two single-stranded oligonucleotide sequences (see below)
are synthesized, annealed, and repetitively filled in, denatured,
and reannealed to form double-stranded oligonucleotides that, when
digested with AflII and BglII, can be cloned into the polylinker of
the expression vector, Cadus 1239. The two single-stranded
oligonucleotides used for the cloning have the following
sequences:
12 5' GG TAC TCG AGT GAA AAG AAG GAC AAC 3' (SEQ ID NO:24) 5' CG
TAC TTA AGC AAT AAC ACA (NNN).sub.n GTT GTC CTT CTT TTC ACT CGA
(SEQ ID NOS:25 and 119) GTA CC 3'
[0361] where N is any chosen nucleotide and n is any chosen
integer. Yeast transformed with the resulting plasmids will
transport--through the pathway normally used for the export of
a-factor--farnesylated, carboxymethylated peptides whose amino acid
sequence is determined by the particular choice of N and n (FIG.
6).
EXAMPLE 4
Peptide Secretion/Transport
[0362] This example demonstrates the ability to engineer yeast such
that they secrete or transport oligonucleotide-encoded peptides (in
this case their pheromones) through the pathways normally used for
the secretion or transport of endogenous pheromones.
[0363] Autocrine MATa Strain CY588
[0364] A MATa strain designed for the expression of peptides in the
context of MF.alpha.1 (i.e., using the MF.alpha.1 expression
vector, Cadus 1215) has been constructed. The genotype of this
strain, which we designate CY588, is MATa bar1 far1-1 fus1-HIS3
ste14::TRP1 ura3 trp1 leu2 his3. The bar1 mutation eliminates the
strain's ability to produce a protease that degrades .alpha.-factor
and that may degrade some peptides encoded by the cloned
oligonucleotides; the far1 mutation abrogates the arrest of growth
which normally follows stimulation of the pheromone response
pathway; an integrated FUS1-HIS3 hybrid gene provides a selectable
signal of activation of the pheromone response pathway; and,
finally, the ste14 mutation lowers background of the FUS1-HIS3
readout. The enzymes responsible for processing of the MF.alpha.1
precursor in MAT.alpha. cells are also expressed in MATa cells
(Sprague and Thorner, in The Molecular and Cellular Biology of the
Yeast Saccharomyces: Gene Expression, 1992, Cold Spring Harbor
Press), therefore, CY588 cells should be able to secrete peptides
encoded by oligonucleotides expressed from plasmid Cadus 1215.
[0365] A high transforming version (tbtl-1) of CY588 was obtained
by crossing CY1013 (CY588 containing an episomal copy of the STE14
gene) (MATa bar1::hisGfar1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2
his3 [STE14 URA3 CEN4]) to CY793 (MAT.alpha. tbtl-1 ura3 leu2 trp1
his3 fus1-HIS2 can1 ste114::TRP1 [FUS1 LEU2 2 .mu.]) and selecting
from the resultant spores a strain possessing the same salient
genotype described for CY588 (see above), and in addition the tb1-1
allele, which confers the capacity for very high efficiency
transformation by electroporation. The selected strain is CY1455
(MATabar1::hisGfar1-1 fus1-HIS3 ste14::TRP1 tbt-1 ura3 trp1 leu2
his3).
[0366] Secretion of peptides in the context of yeast
.alpha.-factor: Experiments were performed to test: 1. the ability
of Cadus 1215 to function as a vector for the expression of
peptides encoded by synthetic oligonucleotides; 2. the suitability
of the oligonucleotides, as designed, to direct the secretion of
peptides through the .alpha.-factor secretory pathway; 3. the
capacity of CY588 to secrete those peptides; and 4. the ability of
CY588 to respond to those peptides that stimulate the pheromone
response pathway by growing on selective media. These experiments
were performed using an oligonucleotide which encodes the 13 amino
acid .alpha.-factor; i.e., the degenerate sequence (NNN).sub.n in
the oligonucleotide cloned into Cadus 1215 (see above) was
specified (n=13) to encode this pheromone. CY588 was transformed
with the resulting plasmid (Cadus 1219), and transformants selected
on uracil-deficient medium were transferred to histidine-deficient
medium supplemented with a range of concentrations of aminotriazole
(an inhibitor of the HIS3 gene product that serves to reduce
background growth). The results, shown in FIG. 7, demonstrate that
the synthetic oligonucleotide, expressed in the context of
MF.alpha.1 by Cadus 1215, conferred upon CY588 an ability to grow
on histidine-deficient media supplemented with aminotriazole. In
summation, these data indicate that: 1. CY588 is competent for the
secretion of a peptide encoded by the (NNN).sub.n sequence of the
synthetic oligonucleotide cloned into and expressed from Cadus
1215; and 2. CY588 can, in an autocrine fashion, respond to a
secreted peptide which stimulates its pheromone response pathway,
in this case by .alpha.-factor binding to STE2.
[0367] Additional experiments were performed to test the utility of
autocrine yeast strains in identifying agonists of the Ste2
receptor from among members of two semi-random .alpha.-factor
libraries, .alpha.-Mid-5 and MF.alpha.-8.
[0368] .alpha.-Mid-5 Library
[0369] A library of semi-random peptides, termed the .alpha.-Mid-5
library, was constructed. In this library, the N-terminal four
amino acids and the C-terminal four amino acids of a 13 residue
peptide are identical to those of native .alpha.-factor while the
central five residues (residues 5-9) are encoded by the degenerate
sequence (NNQ).sub.5. The following oligonucleotides were used in
the construction of the .alpha.-Mid-5 library:
[0370] (1)MF.alpha.Mbo, a 17 mer:
13 (1)MF.alpha.Mbo, a 17 mer: 5' GCGTCACAGACTGATCA (SEQ ID NO:40)
(2)MID5ALF, a 71 mer: 5'
GCCGTCAGTAAAGCTTGGCATTGCTTGNNQNNQNNQNNQMMQCAGCCTATGTACTGATC (SEQ ID
NO:41) AGTCTGTGACGC
[0371] Sequenase (United States Biochemical Corporation, Cleveland,
Ohio) was used to complete the duplex formed after annealing
MF.alpha.Mbo to the MID5ALF oligonucleotide. In the MID5ALF
sequence, N indicates a mixture of A, C, G, and T at ratios of
0.8:1:1.3:1; Q indicates a mixture of C and G at a ratio of 1:1.3.
These ratios were employed to compensate for the different coupling
efficiences of the bases during oligonucleotide synthesis and were
thus intended to normalize the appearance of all bases in the
library. In the oligonucleotide sequences above, the HindIII site
is underlined and the MboI site is emboldened. The double-stranded
oligonucleotide was restricted with HindIII and MboI and ligated to
Cadus 1625 (see above); Cadus 1625 had been prepared to accept the
semi-random oligonucleotides by restriction with HindIII and
BglII.
[0372] The apparant complexity of the .alpha.Mid-5 library is
1.times.10.sup.7. This complexity is based on the number of
bacterial transformants obtained with the library DNA versus
transformants obtained with control vector DNA that lacks insert.
Sequence analysis of six clones from the library demonstrated that
each contained a unique insert.
[0373] To identify peptide members of the .alpha.-Mid-5 library
that could act as agonists on the STE2 receptor, CY1455, a high
transforming version of CY588, was electroporated to enhance uptake
of .alpha.-Mid-5 DNA. Transformants were selected on
uracil-deficient (-Ura) synthetic complete medium and were
transferred to histidine-deficient (-His) synthetic complete medium
supplemented with 0.5 mM or 1 mM aminotriazole.
[0374] Yeast able to grow on -His+aminotriazole medium include (1)
yeast which are dependent on the expression of an .alpha.-factor
variant agonist and (2) yeast which contain mutations that result
in constitutive signalling along the pheromone pathway. Yeast
expressing and secreting a variant STE2 receptor agonist have the
ability to stimulate the growth on -His medium of surrounding CY
1455 cells that do not express such an agonist. Thus a recognizable
formation (termed a "starburst") results, consisting of a central
colony, growing by virtue of autocrine stimuation of the pheromone
pathway, surrounded by satellite colonies, growing by virtue of
paracrine stimulation of the pheromone pathway by the agonist
peptide as that ptpetide diffuses radially fromn the central,
secreting colony.
[0375] In order to identify the peptide sequence responsible for
this "starburst" phenomenon, yeast were transferred from the center
of the "starburst" and streaks were made on -Ura medium to obtain
single colonies. Individual clones from -Ura were tested for the
His.sup.+ phenotype on -His+aminotriazole plates containing a
sparse lawn of CY1455 cells. Autocrine yeast expressing a peptide
agonist exhibited the "starburst" phenotype as the secreted agonist
stimulated the growth of surrounding cells that lacked the peptide
but were capable of responding to it. Constitutive pheromone
pathway mutants were capable of growth on -His+aminotriazole but
were incapable of enabling the grwoth of surrounding lawn
cells.
[0376] Alternatively, streaks of candidate autocrine yeast clones
were made on plates containing 5-fluoroorotic acid (FOA) to obtain
Ura segregants were retested on -His+aminotriazole for the loss of
the His.sup.+ phenotype. Clones that lost the ability to grow on
-His+aminotriazole after selection on FOA (and loss of the
peptide-encoding plasmid) derived from candidate expressors of a
peptide agonist. The plasmid was rescued from candidate clones and
the peptide sequences determined. In addition, a plasmid encoding a
putative Ste2 agonist was reintroduced into CY1455 to confirm that
the presence of the plasmid encoding the peptide agonist conferred
the His.sup.+ phenotype to CY1455. By following the above protocol
novel Ste2 agonists have been identified from the .alpha.-Mid-5
library. Sequences of nine agonists follow, preceded by the
sequence fo the native .alpha.-factor pheromone and by the
oligonucleotide used to encode the native pheromone in these
experiments. (Note the variant codons used in the
.alpha.-factor-encoding oligonucleotide for glutamine and proline
in the C-terminal amino acids of .alpha.-factor). Below each
nucleotide 35 sequence is the encoded amino acid sequence with
variations from the native-pheromone underlined.
14 .alpha.-factor TGG CAT TGG TTG CAG CTA AAA CCT GGC CAA CCA ATG
TAC encodes Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr
(DNA, SEQ ID NO:42, AAS, SEQ ID NO:43) .alpha.-factor oligo: TGG
CAT TGG TTG CAG CTA AAA CCT GGC CAG CCT ATG TAC encodes Trp His Trp
Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr M1 TGG CAT TGG TTG TCC TTG
TCG CCC GGG CAG CCT ATG TAC encodes Trp His Trp Leu Ser Leu Ser Pro
Gly Gln Pro Met Tyr M2 TGG CAT TGG TTG TCC CTG GAC GCT GGC CAG CCT
ATG TAC encodes Trp His Trp Leu Ser Leu Asp Ala Gly Gln Pro Met Tyr
M3 TGG CAT TGG TTG ACC TTG ATG GCC GGG CAG CCT ATG TAC encodes Trp
His Trp Leu Thr Leu Met Ala Gly Gln Pro Met Tyr M4 TGG CAT TGG TTG
CAG CTG TCG GCG GGC CAG CCT ATG TAC encodes Trp His Trp Leu Gln Leu
Ser Ala Gly Gln Pro Met Tyr M5 TGG CAT TGG TTG AGG TTG CAG TCC GGC
CAG CCT ATG TAC encodes Trp His Trp Leu Arg Leu Gln Ser Gly Gln Pro
Met Tyr M6 TGG CAT TGG TTG CGC TTG TCC GCC GGG CAG CCT ATG TAC
encodes Trp His Trp Leu Arg Leu Ser Ala Gly Gln Pro Met Tyr M7 TGG
CAT TGG TTG TCG CTC GTC CCG GGG CAG CCT ATG TAC encodes Trp His Trp
Leu Ser Leu Val Pro Gly Gln Pro Met Tyr M8 TGG CAT TGG TTG TCC CTG
TAC CCC GGG CAG CCT ATG TAC encodes Trp His Trp Leu Ser Leu Tyr Pro
Gly Gln Pro Met Tyr M9 TGG CAT TGG TTG CGG CTG CAG CCC GGG CAG CCT
ATG TAC encodes Trp His Trp Leu Arg Leu Gln Pro Gly Gln Pro Met
Tyr
[0377] The nine peptide agonists of the Ste2 receptor above were
derived from one electroporation of CY1455 using 1 .mu.g of the
.alpha.-Mid-5 library DNA. Approximately 3.times.10.sup.5
transformants were obtained, representing approximately 0.03% of
the sequences present in that library.
[0378] MF.alpha.-8 Library
[0379] A semi-random .alpha.-factor library was obtained through
synthesis of mutagenized .alpha.-factor oligonucleotides such that
1 in 10,000 peptide products were expected to be genuine
.alpha.-factor. The mutagenesis was accomplished with doped
synthesis of the oligonucleotides: each nucleotide was made
approximately 68% accurate by synthesizing the following two
oligos:
[0380] 5'CTGGATGCGA AGACTCAGCT (20 mer) (oligo060) (SEQ ID NO:44)
where the FokI site is underlined and the BbsI site is
emboldened.
[0381] 5'CGGATGATCA gta cat tgg ttg gcc agg ttt tag ctg caa cca atg
cca AGC TGA GTC TTC GCA TCC AG (69 mer) (oligo074) (SEQ ID
NO:45)
[0382] where the BclI site is italicized, the FokI site is
underlined, the BbsI site is emboldened. The lower case letters
indicate a mixture of 67% of that nucleotide and 11% of each of the
other three nucleotides (e.g. t indicates 67% T and 11% A, 11% C,
and 11% G). Note that digestion of the double-stranded
oligonucleotide by FokI or BbsI will yield an identical 5' end that
is compatible with HindIII ends. Oligos 060 and 074 will form the
following double-stranded molecule when annealed:
15 5' CTGGATGCGAAGACTCAGCT 3' GACCTACGCTTCTGAGTCGA acc gta acc aac
gtc gat ttt gga ccg gtt ggt tac atg ACTAGTAGGC 5'
[0383] The duplex was repetitively filled-in using Taq DNA
polymerase (Promega Corporation, Madison, Wis.). The
double-stranded product was restricted with BbsI and BclI and
ligated into HindIII- and BglII-digested Cadus 1373. The BglII/BclI
joint creates a TGA stop codon for the termination of translation
of the randomers. Using this approach, the MF.alpha.-5.8 library (a
library of apparent low complexity based on PCE analysis of
oligonucleotide insert frequency) was constructed. To identify
peptide members of the MF.alpha.-5.8 library that could act as
agonists on the STE2 receptor, CY1455, a high transforming version
of CY588, was electroporated to enhance uptake of MF.alpha.-5.8
DNA. Transformants were selected on uracil-deficient (-Ura)
synthetic complete medium and were transferred to
histidine-deficient (-His) synthetic complete medium supplemented
with 1.0 mM or 2.5 mM aminotriazole. Yeast from colonies which were
surrounded by satellite growth were transferred as streaks to -Ura
medium to obtain single colonies. Yeast from single colonies wree
then tested for the His.sup.+ phenotype on -His+aminotriazole
plates. Sequence analysis of seven of the plasmids rescued from
His.sup.+ yeast revealed three unique .alpha.-factor variants that
acted as agonists on the STE2 receptor.
[0384] 1.4 independent clones had the following sequence:
16 TGG CAT TGG CTA CAG CTA ACG CCT GGG CAA CCA ATG TAC (SEQ ID
NO:46) encoding Trp His Trp Leu Gln Leu Thr Pro Gly Gln Pro Met Tyr
(SEQ ID NO:47) 2. 2 independent clones had the following sequence:
TGG CAT TGG CTG GAG CTT ATG CCT GGC CAA CCA TTA TAC (SEQ ID NO:48)
encoding Trp His Trp Leu Glu Leu Met Pro Gly Gln Pro Leu Tyr (SEQ
ID NO:49) 3. TGG CAT TGG ATG GAG OTA AGA CCT GGC CAA CCA ATG TAC
(SEQ ID NO:50) encoding Trp His Trp Met Glu Leu Arg Pro Gly Gln Pro
Met Tyr (SEQ ID NO:51)
[0385] Autocrine Mata Strain CY599
[0386] A MATa strain designed for the expression of peptides in the
context of MFA1 (i.e., using the MFA1 expression vector, Cadus
1239) has been constructed. The genotype of this strain, designated
CY599, is MATa mfa1 mfa2 far1-1 his3::fus1-HIS3 ste2-STE3 ura3 met1
ade1 leu2. In this strain, Cadus 1147 (see above) was used to
replace STE2 with a hybrid gene in which the STE3 coding region is
under the control of expression elements which normally drive the
expression of STE2. As a result, the a-factor receptor replaces the
.alpha.-factor receptor. The genes which encode a-factor are
deleted from this strain; the far1 mutation abrogates the arrest of
growth which normally follows stimulation of the pheromone response
pathway; and the FUS1-HIS3 hybrid gene (integrated at the HIS3
locus) provides a selectable signal of activation of the pheromone
response pathway. CY599 cells were expected to be capable of the
transport of a-factor or a-factor-like peptides encoded by
oligonucleotides expressed from Cadus 1239 by virtue of expression
of the endogenous yeast transporter, Ste6.
[0387] Transport of Peptides by the Yeast a-factor Pathway
[0388] Experiments were performed to test: 1. the ability of Cadus
1239 to function as a vector for the expression of peptides encoded
by synthetic oligonucleotides; 2. the suitability of the
oligonucleotides, as designed, to direct the export of
farnesylated, carboxymethylated peptides through the pathway
normally used by a-factor; 3. the capacity of CY599 to export these
peptides; and 4. the ability of CY599 to respond to those peptides
that stimulate the pheromone response pathway by growing on
selective media. These tests were performed using an
oligonucleotide which encodes the 12 amino acid a-factor;
specifically, the degenerate sequence (NNN).sub.n in the
oligonucleotide cloned into Cadus 1239 (see above) (with n=12)
encodes the peptide component of a-factor pheromone. CY599 was
transformed with the resulting plasmid (Cadus 1220), and
transformants selected on uracil-deficient medium were transferred
to histidine-deficient medium supplemented with a range of
concentrations of aminotriazole. The results, shown in FIG. 8,
demonstrate that the synthetic oligonucleotide, expressed in the
context of MFA1 by Cadus 1220, conferred upon CY599 enhanced
aminotriazole-resistant growth on histidine-deficient media. In
summation, these data indicate: 1. Cadus 1220 and the designed
oligonucleotide are competent to direct the expression and export
of a farnesylated, carboxymethylated peptide encoded by the
(NNN).sub.n sequence of the synthetic oligonucleotide; and 2. CY599
can, in an autocrine fashion, respond to a farnesylated,
carboxymethylated peptide that stimulates its pheromone response
pathway, in this case signaling initiates as a-factor binds to
STE3.
EXAMPLE 5
Proof of Concept
[0389] This example will demonstrate the utility of the autocrine
system for the discovery of peptides which behave as functional
pheromone analogues. By analogy, this system can be used to
discover peptides that productively interact with any pheromone
receptor surrogates.
[0390] CY588 (see strain 5, Example 2 above) will be transformed
with CADUS 1215 containing oligonucleotides encoding random
tridecapeptides for the isolation of functional .alpha.-factor
analogues (FIG. 4). CY599 (see strain 6, Example 2 above) will be
transformed with CADUS 1239 containing oligos of random sequence
for the isolation of functional a-factor analogues (FIG. 6).
Colonies of either strain which can grow on histidine-deficient
media following transformation will be expanded for the preparation
of plasmid DNA, and the oligonucleotide cloned into the expression
plasmid will be sequenced to determine the amino acid sequence of
the peptide which presumably activates the pheromone receptor. This
plasmid will then be transfected into an isogenic strain to confirm
its ability to encode a peptide which activates the pheromone
receptor. Successful completion of these experiments will
demonstrate the potential of the system for the discovery of
peptides which can activate membrane receptors coupled to the
pheromone response pathway.
[0391] Random oligonucleotides to be expressed by the expression
plasmid CADUS 1215 will encode tridecapeptides constructed as
5'CGTGAAGCTTAAGCGTGAGGCAGAAGCT(NNK).sub.13TGATCATCCG, (SEQ ID NO:6)
where N is any nucleotide, K is either T or G at a ratio of 40:60
(see Proc Natl Acad Sci 87:6378, 1990; ibid 89:5393, 1992), and the
AflII and BclI sites are underlined. This oligonucleotide is
designed such that: the AflII and BclI sites permit inserting the
oligos into the AflII and BglII site of CADUS 1215 (see FIG. 4);
the HindIII site just 5' to the AflII site in the 5' end of the
oligo allows future flexibility with cloning of the oligos; the
virtual repeat of GAGGCT and the GAGA repeats which are present in
the wild-type sequence and which can form triple helixes are
changed without altering the encoded amino acids. The random
oligonucleotides described above will actually be constructed from
the following two oligos:
17 5' CGTGAAGCTTAAGCGTGAGGCAGAAGCT (SEQ ID NO:26) and 5'
CGGATGATCA(MNN).sub.13AGCTTCTG (SEQ ID NO:27),
[0392] where M is either A or C at a ratio of 40:60. The oligos
will be annealed with one another and repetitively filled in,
denatured, and reannealed (Kay et al, Gene, 1993). The
double-stranded product will be cut with AflII and BclI and ligated
into the AflII- and BglII-digested CADUS 1215. The BglII/BclI joint
will create a TGA stop codon for termination of translation of the
randomers (FIG. 4). Because of the TA content of the Afl overhang,
the oligos will be ligated to the AflII-and BglII-digested
pADC-MF.alpha. at 4.degree. C.
[0393] Random oligonucleotides to be expressed by the expression
plasmid CADUS 1239 will encode monodecapeptides constructed as
5'GGTACTCGAGTGAAAAGAAGGACAAC(NNK).sub.11TGTGTTATTGCTTAAGTACG (SEQ
ID NO:12),
[0394] where N is any nucleotide, K is either T or G at a ratio of
40:60 (see Proc Natl Acad set 87:6378, 1990; ibid 89:5393, 1992),
and the XhoI and AflII sites are underlined. When cloned into the
XhoI and AflII sites of CADUS 1239 the propeptides expressed under
the control of the ADH1 promoter will contain the entire leader
peptide of MFa1, followed by 11 random amino acids, followed by
triplets encoding CVIA (the C-terminal tetrapeptide of wild-type
a-factor). Processing of the propeptide should result in the
secretion of dodecapeptides which contain 11 random amino acids
followed by a C-terminal, farnesylated, carboxymethylated
cysteine.
[0395] Using the procedure described above, the oligonucleotides
for expression in CADUS 1239 will actually be constructed from the
following two oligos:
18 5' GGTACTCGAGTGAAAAGAAGGACAAC (SEQ ID NO:28) and 5'
CGTACTTAAGCAATAACAca (MNN).sub.11GTTGTCC (SEQ ID NO:29),
[0396] where M is either A or C at a ratio of 40:60, and the XhoI
and AflII sites are underlined.
[0397] Discovery of a-factor Analogues from a Random Peptide
Library
[0398] An optimized version of strain 6 (Example 2 above) was
derived. This yeast strain, CY2012 (MATa ste2-STE3 far1.DELTA.1442
mfa1::LEU2 mfa2-lacZ fus1-HIS3 tbt1-1 ura3 leu2 his3 trp1 suc2),
was constructed as follows. From a cross of CY570 (MATa mfa1::LEU2
mfa2-lacZ ura3 trp1 his3.DELTA.200 can1 leu2 fus1-HIS3 [MFA1 URA3
2.mu.] [FUS1.DELTA.8-73 TRP1 CEN6]) by CY1624 (MAT.alpha. tbt1-1
fus1-HIS3 trp1 ura3 leu2 his3 lys2-801 SUC+), a spore was selected
(CY1877) of the following genotype: MATa mfa1::LEU2 mfa2-lacZ
fus1-HIS3 tbt1-1 ura3 leu2 his3 trp1 suc2. This strain lacks both
genes (MFA1 and MFA2) encoding a-factor precursors, contains the
appropriate pheromone pathway reporter gene (fus1-HIS3), and
transforms by electroporation at high efficiency (tbt1-1). This
strain was altered by deletion of the FAR1 gene (with Cadus 1442;
see Example 6), and replacement of STE2 coding sequences with that
of STE3 (see Example 1) to yield CY2012.
[0399] This strain was transformed with plasmid DNA from a random
a-factor library by electroporation and plated on 17 synthetic
complete plates lacking uracil (-Ura), yielding approximately
10.sup.5 Ura.sup.+ colonies per plate after 2 days at 30.degree. C.
These colonies were replica plated to histidine-deficient synthetic
complete media (-His) containing 0.2 mM 3-aminotriazole and after
three days at 30.degree. C. 35 His.sup.+ replicas were streaked to
-Ura plates. The resultant colonies, 3 from each isolate, were
retested for their His.sup.+ phenotype, and streaked to
5-fluoroorotic acid plates to obtain Ura.sup.- segregants (lacking
a library plasmid). Those Ura.sup.- segregants were tested for the
loss of their His.sup.+ phenotype. Ten of the original isolates
passed these tests; in two cases only one of the three Ura.sup.+
colonies purified from the isolate retained the His.sup.+
phenotype, but nevertheless subsequently segregated Ura.sup.-
His.sup.- colonies.
[0400] A single plasmid (corresponding to a bacterial colony) was
obtained from each of the ten isolates, and reintroduced into
CY2012. Eight of the ten plasmids passed the test of retaining the
ability to confer the His.sup.+ phenotype on CY2012 (the two that
failed correspond to the two isolates that were mentioned above,
suggesting that these isolates contain at least one "irrelevant"
plasmid). Sequencing of the randomized insert in the eight plasmids
of interest revealed that four contain the sequence:
19 TAT GCT CTG TTT GTT CAT TTT TTT GAT ATT CCG (SEQ ID NO:52) Tyr
Ala Leu Phe Val His Phe Phe Asp Ile Pro, (SEQ ID NO:53)
[0401] two contain the sequence:
20 TTT AAG GGT CAG GTG CGT TTT GTG GTT CTT GCT (SEQ ID NO:54) Phe
Lys Gly Gln Val Arg Phe Val Val Leu Ala, (SEQ ID NO:55)
[0402] and two contain the sequence:
21 CTT ATG TCT CCG TCT TTT TTT TTT TTG CCT GCG (SEQ ID NO:56) Leu
Met Ser Pro Ser Phe Phe Phe Leu Pro Ala (SEQ ID NO:57)
[0403] Clearly, these sequences encode novel peptides, as the
native a-factor sequence differs considerably: Tyr Ile Ile Lys Gly
Val Phe Trp Asp Pro Ala.
[0404] The a-factor variants identified from random peptide
libraries have utility as "improved" substrates of ABC is
transporters expressed in yeast. For example, identification of a
preferred substrate of human MDR, one that retains agonist activity
on the pheromone receptor, would permit the establishment of robust
yeast screens to be used in the discovery of compounds that affect
transporter function.
EXAMPLE 6
Drug Screens Designed to Permit Discovery of Molecules Which
Modulate the Function of ATP-dependent Transmembrane
Transporters
[0405] The availability of cloned DNA encoding the related proteins
human Mdr1, human CFTR and human MRP, will allow the construction
of yeast strains expressing these molecules. The resultant strains
will be essential to the design of microbiological assays which can
be used to probe the function of these proteins and to discover
molecules capable of inhibiting or enhancing their function in
cellular resistance to chemotherapeutics or in ion transport. The
present assay makes use of the transport of the yeast mating
pheromone a-factor from autocrine yeast expressing a human protein
capable of substituting for yeast Ste6.
[0406] A. MFa1- and Mdr1-containing Plasmids Obtained From Karl
Kuchler (University of Vienna) for Use in These Experiments:
[0407] (1) pYMA177 (denoted Cadus 1067), see FIG. 9.
[0408] (2) pKK1 includes sequence encoding MFa1 in YEp351; a-factor
is overexpressed from this plasmid due to increased plasmid copy
number
[0409] (3) pHaMDR1(wt) provides wild type Mdr1 cDNA in a retroviral
vector (initially obtained from Michael Gottesman, NIH).
[0410] B. Plasmids Constructed at Cadus for Use in These
Experiments
[0411] A 1.5 kb BamHI-BglII fragment which includes MFa1 sequence
was derived from pYMA177 and ligated to BamHI-digested pYMA177 to
yield Cadus 1079. Cadus 1079 was digested with BglII and
recircularized to delete a 950 bp fragment containing sequence
encoding the G185V mutant of human Mdr1; the resultant plasmid is
Cadus 1093. pHaMDR1 was digested with BglII to allow isolation of a
965 bp fragment containing wild type human Mdr1 (G185) sequence.
The 965 bp BglII fragment was inserted into BglII-digested Cadus
1093 to yield Cadus 1097. The Cadus 1097 construct was verified by
sequencing using dideoxy nucleotides. To yield Cadus 1164, pYMA177
was digested with BamHI and recircularized to eliminate the
sequence encoding MFa1. Cadus 1165 was constructed by ligating a
700 bp BglII-BamHI fragment from pYMA177 to the large BamHI to
BglII fragment of pYMA177. This results in the removal of both the
1.6 kb MFa BamHI fragment and the 965 bp BglII fragment encoding
human Mdr1 (G185V); the resulting plasmid is Cadus 1165. Cadus 1176
resulted from the ligation of a 965 bp BglII fragment from pHaMDR1,
containing sequence encoding wild type human Mdr1, to
BglII-digested Cadus 1165. pRS426 (Cadus 1019) served as a URA3
control plasmid.
[0412] The final plasmid array used in these experiments is as
follows:
22 no mutant Mdr1 wt Mdr1 Mdr1 (G185V) (G185) a-factor 1065 1079
1097 overexpression no a-factor 1019 1164 1176 overexpression
[0413] Evidence for the Expression of Human Mdr1 Constructs in
Yeast
[0414] CADUS plasmids 1065, 1079 and 1097 as well as a URA3 control
plasmid (pRS426=1019) were transformed into a ura3, ste6strain of
yeast (WKK6=CY20=MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1
can1-100 ste6::HIS3, obtained from Karl Kuchler). Individual
transformants were grown overnight in SD-URA media and lawns
containing approximately 5.times.10.sup.6 cells were poured onto
YPD plates in 3 ml of YPD top agar. Sterile filter disks were
placed on the plates after the top agar had solidified, and 5 ml of
DMSO or 5 mM valinomycin in DMSO were spotted onto the filter
disks. By this assay, expression of mutant Mdr1 from plasmid 1079
in WKK6 cells conferred weak resistance to valinomycin while
expression of wild type Mdr1 from plasmid 1097 conferred complete
resistance.
[0415] Attempts to Assay Mdr1 Activity by a-factor Transport in a
Two Cell System
[0416] Several attempts to demonstrate mating of WKK6 transformed
with the various human Mdr1 constructs failed. This is in contrast
to published experiments utilizing the mouse mdr3 gene, in which a
partial complementation of mating deficiency was seen (Raymond et
al., 1992).
[0417] A "halo" assay was performed by patching WKK6 cells
containing the various Mdr1 constructs (1019, 1065, 1079 &
1097) onto a lawn of Mat.alpha. cells which are supersensitive to
a-factor (CY32=MAT.alpha. leu2-3, 112 trp1-289 ura3-52 his3*1
sst2*2 GAL+). Although all halos were much smaller than those
produced by STE6.sup.+ cells, a small difference in halo size was
observed with the relative order 1097>1079.about.1065- >1019.
This indicates that a-factor can be transported by the wild-type
human Mdr1 protein expressed in yeast deleted for STE6, however,
the halo assay does not appear to be amenable to rapid drug
screening.
[0418] The halo assay detects a-factor secreted from ste6.sup.-
Mdr1 strains by growth arrest of cells present in the indicator
lawn. An alternative, and potentially more sensitive, method makes
use of the transcriptional response to pheromone signaling. A
strain with an a-factor responsive HIS3 gene (CY104=MAT.alpha. ura3
leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1) was cross-streaked with
STE6.sup.+ (CY19=W303-1a=MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1
ade2-1 can1-100) or ste6.sup.- (WKK6=CY20=MATa ura3-1 leu2-3, 112
his3-11, 15 trp1-1 ade2-1 can1-100 ste6::HIS3) strains containing
various plasmids. The cross-streaking was performed on a plate
lacking histidine and tryptophan so that only the CY104 indicator
strain would grow if stimulated by a-factor. The order of a-factor
secretion seen by this method was
CY58>CY61.apprxeq.CY62.apprxeq.CY63>CY60 where: CY58=CY19
(STE6.sup.+, 1019), CY60=CY20(ste6.sup.-, 1019), CY61=CY20
(ste6.sup.-, 1065), CY62=CY20 (ste6.sup.-, 1079) and CY63=CY20
(ste6.sup.-, 1097). Thus, the difference in a-factor production
between STE6.sup.+ and ste6.sup.- and between a-factor
overproduction or not is detectable in this system, whereas the
activity of Mdr1 in secreting a-factor is not. It was not clear
from these experiments whether the signal generated from the
ste6.sup.-, a-factor overproducing strains was due to an alternate
pathway for a-factor secretion or the release of a-factor from
lysed cells.
[0419] Assay of Mdr1 Activity by a-factor Transport in an Autocrine
System
[0420] A single strain system for the detection of Mdr1-mediated a
factor transport was constructed in order to improve sensitivity
and reproducibility and to circumvent the potential false signal of
a-factor release from lysed cells (a cell concentration-dependent
phenomenon). A strain was constructed (CY293=MATa ura3 leu2 trp1
his3 fus1-HIS3 can1 ade2-1 ste2-STE3 ste6::TRP1) which could
respond to a-factor by growing on media lacking histidine and in
which the only impediment to the secretion of a-factor is the lack
of a functional STE6 gene. Indeed, the immediate precursor to this
strain, which did contain a functional STE6 gene, was able to grow
vigorously on -HIS media containing 3 mM aminotriazole, whereas
CY293 did not grow at all. The addition of aminotriazole, a
competitive inhibitor of the HIS3 enzyme, is necessary to reduce
the background growth of these strains.
[0421] The Mdr1-containing plasmids were introduced into CY293, and
the transformants were streaked onto -HIS plates containing either
0.1 or 0.33 mM aminotriazole. The growth pattern thus generated was
1097>1067>1065.apprxeq.1176>1164.apprxeq.1019. The
STE6.sup.+ parent of CY293 exhibited a much more vigorous growth
than any of these transformants. However, human Mdr1-mediated
a-factor secretion is clearly detectable in this autocrine system.
CY293 transformed with 1097 can be used to screen for drugs which
enhance the activity of the Mdr1 protein (increased growth on -HIS
+aminotriazole) as well as drugs which inhibit Mdr1 activity
(decreased growth on -HIS +aminotriazole). In the latter case,
controls must be designed to identify compounds which inhibit yeast
growth in an Mdr1-independent fashion.
[0422] Additional strains, with improvements over CY293 (see
example 6 [76-35]) for the expression of mammalian ABC transporters
were constructed. These strains contain the tbt1-1 allele,
conferring high-efficiency transformation by electroporation, and a
lesion in the FAR1 gene. In addition, they are auxotrophic for
tryptophan, and hence can serve as hosts for TRP1-based
plasmids.
[0423] Two starting strains were selected, CY1555 (MATa tbt1-1
fus1-HIS3 trp1 ura3 leu2 his3 lys2-801 SUC+) and CY1557 (MATa
tbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 suc2). A plasmid containing an
internal deletion of FAR1 was constructed by amplifying genomic
sequences corresponding to the 5' end and the 3' end of FAR1, and
ligating them together in pRS406 (an integrative vector containing
the URA3 gene), thereby creating a deletion from the 50th to the
696th predicted codon of FAR1. The oligonucleotides used for
amplification were: for the 5' segment of FAR1:
23 for the 5' segment of FAR1: 5'-CGGGATCCGATGCAATTTTCAACAT- GC-3'
(23FAR1) (SEQ ID NO:58) and 5'-GCTCTAGATGCTACTGATCC- CGC-3'
(1RAF616) (SEQ ID NO:59) and for the 3' segment of FAR1:
5'-CGCCGCATGACTCCATTG-3' (2552FAP1) (SEQ ID NO:60) and
5'-GGGGTACCAATAGGTTCTTTCTTAGG-3' (1RAF2979). (SEQ ID NO:61)
[0424] The resultant amplification products were restricted with
BamHI and XbaI (5' segment; 0.6 kb) or NheI and KpnI (3' segment;
0.4 kb), and ligated into pRS406 (Cadus 1011) that had been
restricted with BamHI and KpnI. The resultant plasmid (Cadus 1442)
was restricted with EcoRI to direct integration at the FAR1 locus,
Ura+ transformants were purified and subjected to selection on
5-fluoro-orotic acid, and Ura-clones were screened for the impaired
mating ability conferred by the far1 deletion. The STE6 gene was
subsequently deleted using the ste6::hisG-URA3 plasmid (Cadus 1170;
constructed by Karl Kuchler), and the STE2 coding sequences were
replaced with STE3 coding sequences as described in Example 1. The
resultant strains were named CY1880 (MATa ste2-STE3 ste6::hisG
far1.DELTA.1442 tbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 suc2) and
CY1882 (MATa ste2-STE3 ste6::hisG far1.DELTA.1442 tbt1-1 fus1-HIS3
trp1 ura3 leu2 his3 lys2-801 SUC+)
[0425] Like strain CY293 these strains show a dramatic enhancement
of growth (via the fus1-HIS3 reporter gene) under conditions of
histidine starvation when transformed with the MDR1-encoding
plasmids Cadus 1097 and Cadus 1176 (as compared to control plasmids
lacking MDR1, Cadus 1065 and 1019). In addition, these strains
display more robust general growth, transform by electroporation at
high efficiency (the tbt1-1 effect), lack susceptibility to
pheromone-induced growth arrest (due to inactivation of FAR1), and
act as hosts for TRP1-based plasmids (ste6::hisG instead of
ste6::TRP1).
[0426] These strains also act as suitable hosts for the STE6-CFTR
chimeras constructed by Teem et al. (1993) (see Example 7). When
compared with CY293 in their ability to distinguish between wild
type and .DELTA.F508 STE6-CFTR in their ability to transport
a-factor, a similar enhancement is seen:
24 Host strain wild type/.DELTA.F508 growth ratio CY293 18 CY1880 7
CY1882 8
[0427] The strains were transformed with Cadus 1515 (STE6-CFTR(H5)
URA3 CEN) or 1516 (STE6-CFTR(H5).DELTA.FS508 URA3 CEN) and
inoculated at various cell densities (OD.sub.600=0.003 to 0.048)
into histidine-free media containing various concentrations of
3-aminotriazole (0 to 1.2 mM). After overnight growth in microtiter
wells the optical densities at 600 nm of the wells were measured,
and ratios for wild type vs. .DELTA.F508 calculated. The ratios
reflect the highest ratios obtained in this experiment but not
necessarily the highest ratio it would be possible to obtain.
[0428] Improvements on the Autocrine Yeast Expressing Mdr1
[0429] The results described above indicate that the human Mdr1
protein transports a-factor less efficiently than does the yeast
STE6 protein. Attempts will be made to isolate mutant a-factor
molecules which are transported more efficiently by human Mdr1 and
yet which retain agonist activity on the a-factor receptor (STE3
protein). To do this, a-factor coding sequences will be chemically
synthesized using "dirty" nucleotides and inserted into an a-factor
expression cassette. An example of "dirty" synthesis would be to
incorporate nucleotide from a mixture of 70% and 10% each of A, T
and C at a position in the a-factor sequence where G would normally
appear. Using oligonucleotides generated in this manner, a diverse
library of peptides can be expressed in yeast and screened to
identify those peptides which retain the ability to signal to the
STE3 protein but which are also a favorable substrate for transport
by human Mdr1.
[0430] A second improvement to the system will be the addition of a
pheromone-inducible "negative selection" marker. For instance, the
FUS1 promoter can be connected to GAL1 coding sequences. Expression
of GAL1 is toxic in the presence of galactose in strains which
contain mutations in either the GAL7 or GAL10 genes. In the context
of an autocrine Mdr1 strain, this selection system should render
cells galactose-sensitive. Addition of a compound which inhibits
the ability of the Mdr1 protein to secrete a-factor would allow
this strain to grow on galactose-containing media. This selection
system would also eliminate false positives due to lethality.
Controls must still be designed to identify compounds which
interfere at other points in the pheromone response pathway.
[0431] The third improvement in the system involves inactivation of
yeast genes which function equivalently to mammalian MDR genes. A
network of genes involved in pleiotropic drug resistance (PDR) have
been identified in yeast. This modification will be useful in any
yeast screen designed to assay the interaction of compounds with
intracellular targets. The improved autocrine yeast strain,
expressing human Mdr1, will be used to screen compound libraries
for molecules which inhibit the transport function of this protein.
In addition, Mf.alpha. expression cassettes containing
oligonucleotides encoding random peptides will be expressed in the
autocrine Mdr1 strain to identify peptides capable of inhibiting
the transport of a-factor, or an a-factor analogue by Mdr1.
EXAMPLE 7
Identification of an Analogue of Yeast a-factor That is Transported
by Wild Type Human CFTR
[0432] This example describes the use of autocrine yeast strains to
identify molecules capable of enhancing transport by dysfunctional
ATP-dependent transmembrane transporters, e.g. mutant human CFTR
proteins. The wild type human CFTR protein will not substitute for
Ste6 function in yeast by transporting native a-factor pheromone
(John Teem, unpublished observations). In order to maximally
exploit autocrine yeast strains for the discovery of molecules
which enhance mutant CFTR function, an a-factor-like peptide which
can serve as a substrate for transport by CFTR will be identified.
An a-factor analogue must also bind functionally to the pheromone
receptor, Ste3, in order to initiate pheromone signalling. The CFTR
transport substrate will be identified by expressing a randomly
mutated MFa expression cassette in autocrine yeast deleted for Ste6
expression, but expressing the wild type human CFTR protein.
[0433] Expression of Mutant Human CFTR Proteins in Autocrine
Yeast
[0434] Transport of an a-factor-like peptide pheromone by CFTR will
serve as the basis for the design of screens to be used in the
identification of compounds which augment the transport function of
CFTR proteins containing cystic fibrosis (CF) mutations. Based on
studies done by Teem et al. (1993) using Ste6/CFTR chimeras, mutant
CFTR is not expected to efficiently transport an a-factor analogue.
Teem et al. (1993) have made chimeric proteins by substituting
varying portions of the sequences encoding the first nucleotide
binding domain of human CFTR for analogous sequence in yeast STE6.
The chimeric proteins will transport native yeast a-factor when
expressed in yeast. However, introduction of a CF mutation
(.DELTA.F508) reduces the ability of these proteins to transport
the yeast pheromone. Teem et al. have also identified revertants in
yeast, i.e., proteins containing second site mutations which
restore the ability of chimeras bearing the CF mutation to
transport a-factor. Introduction of the revertant mutations into
defective CFTR protein expressed in mammalian cells decreased in
part the processing and channel defects of the .DELTA.F508 protein.
With the identification of an analogue of yeast a-factor that will
serve as a substrate for transport by CFTR, mutations can be
introduced directly into the human CFTR protein expressed in yeast,
eliminating the necessity of creating chimeric proteins containing
Ste6 sequence. This will permit the targeting of potential CF
therapeutics to the entire human CFTR molecule. Mutations of
interest include G551D, N1303K and .DELTA.I507 in addition to
.DELTA.F508; these mutations are naturally occurring, give rise to
cystic fibrosis in affected individuals, and appear to affect
either the transport and processing of CFTR or the regulation of
function of that protein (Welsh and Smith 1993). These mutations
also rank among the most common alterations of CFTR thus far
identified in CF patients. If a peptide which will serve as a
substrate for transport by CFTR cannot be identified, chimeric
Ste6/CFTR proteins and unaltered a-factor can be utilized in
screens based on autocrine strains of yeast. These strains offer
distinct advantages in screening applications: easy adaptability to
large-scale automation, simplicity and increased sensitivity when
compared to traditional yeast mating cell assays of pheromone
signaling, and the potential to employ the instant technology to
identify active peptide structures.
[0435] Identification of Compounds That Enhance Transport of
a-factor-like Peptide by Mutant CFTR
[0436] Autocrine strains expressing mutant human CFTR protein will
be capable of growth on rich media but, due to inadequate transport
of and signaling by an a-factor analogue, will not grow efficiently
on selective (histidine-deficient) media. Pheromone signaling in
these strains initiates expression from a FUS1 promoter sequence
controlling transcription of the His3 enzyme; expression of His3 is
required for growth on media lacking histidine. These strains will
be used to screen compound libraries to identify molecules which
reverse the inability of the mutant CFTR to transport a-factor
analogue and permit growth of the yeast on histidine-deficient
medium. Alternatively, active compounds would be capable of
signaling to the a-factor receptor, Ste3, directly, or may interact
with the pheromone response pathway elsewhere. Suitable controls
would differentiate among these possibilities.
[0437] Identification of Random Peptides That Enhance Transport by
Mutant CFTR
[0438] Plasmids containing oligonucleotides which encode random
peptides will be expressed in autocrine yeast bearing a mutant
human CFTR. These peptides, expressed using .alpha.-factor-based
expression cassettes, will be transported to the extracellular
environment via the yeast secretory pathway. Peptides of interest
will be identified by virtue of their ability to permit the growth
of a mutant CFTR-containing strain on histidine-deficient medium.
Active peptides would permit transport of a-factor analogue by the
mutant human CFTR. Alternatively, a peptide may interact at other
points along the pheromone response pathway. Suitable controls
would differentiate between these possible outcomes.
EXAMPLE 8
Prophetic Example of Substitution of Prohormone Convertase PC1 for
Yeast KEX2
[0439] The mammalian prohormone convertases PC1/PC3 and PC2 are
involved in the proteolytic processing of proopiomelanocortin
(POMC), with PC1 preferentially releasing adrenocorticotropic
hormone (ACTH) and .beta.-lipotropin and PC2 preferentially
releasing .beta.-endorphin, N-terminally extended ACTH containing
the joining peptide (JP), and either .alpha.-melanocyte-stimulating
hormone (.alpha.-MSH) or des-acetyl .alpha.-MSH (Benjannet, S., et
al. (1991) Proc. Natl. Acad. Sci. USA 88:3564-3568; Seideh N. G. et
al. (1992) FEBS Lett. 310, 235-239). By way of example, a yeast
strain is described in which mammalian PC1 processes a chimeric
pre-pro-POMC/.alpha.-factor peptide, permitting the secretion of
mature .alpha.-factor and stimulation of the screening strain in an
autocrine fashion to histidine protrotrophy. Autocrine strains will
be constructed in which: 1. yeast KEX2 is disrupted; 2. yeast KEX2
activity is substituted with that of mammalian PC1; 3. a novel
MF.alpha. construct containing the dibasic cleavage site recognized
by PC1 (in place of the KEX2 recognition sequence) will be
expressed; 4. production of mature .alpha.-factor will require PC1
activity; and 5. growth of the strain will be stimulated by the
production of mature .alpha.-factor.
[0440] The genotype of the parental strain for this example is MATa
bar1::hisG far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3.
Initially, the KEX2 allele of this strain will be disrupted using
an integrating plasmid (pkex2.DELTA.) encoding the flanking regions
of the KEX2 locus with the entire coding regions from the initiator
codon to the terminator codon deleted. Cleavage of pkex2.DELTA._
with Bsu36I followed by transfection into the parental strain will
result in integration of this plasmid into the KEX2 locus.
Transformation can be scored as conversion to uracil prototrophy.
Subsequent transfer of URA+ transformants to plates containing
5-fluoroorotic acid results in the growth of colonies with the
kex1.DELTA._ allele. Integration can be confirmed by Southern blot
analysis and by colony PCR using oligonucleotide primers flanking
the KEX2 locus. This kex1.DELTA._ strain will be able to grow on
histidine-deficient media in the presence of exogenenously added
.alpha.-factor, but will not be able to grow in an autocrine
fashion on histidine-deficient media once transfected with Cadus
1219 (URA3 2mu-ori REP3 AmpR f1-ori .alpha.-factor) since
processing of the pre-pro-POMC/.alpha.-factor chimera expressed
from Cadus 1219 requires KEX2 activity.
[0441] In the screening strain, PC1 activity will substitute for
the deleted KEX2 activity in the maturation of .alpha.-factor
peptide. PC1 cDNA (accession #M69196) obtained from mouse (Korner
et al (1991) Proc. Natl. Acad. Sci USA 88:6834-6838) was found to
encode a protein of 753 amino acids. Sequences encoding PC1 will be
cloned into a high copy replicating plasmid (Cadus 1289). Yeast
cells transformed with this plasmid will acquire the ability to
grow on leucine-deficient media and will express high levels of PC1
protein due to the presence of the PGK promotor.
[0442] A hybrid gene encoding the prepro-region of human POMC
(accession #K02406; Takahashi, H., et al (1983) Nucleic Acids
Research 11:6847-6858) and the coding region of a single repeat of
mature .alpha.-factor will be constructed in the following fashion.
The prepro-region of human POMC will be amplified with an HindIII
site at the 5' end and a BbsI site at the 3' end using VENT
polymerase and the following primers: 5'GGGAAGCTT
ATGCCGAGATCGTGCTGCCAGCCGC3' (SEQ ID NO:30) (HindIII site is
underlined and initiation codon is italic bold) and antisense
5'GGGGAAGACTTCTGCCCTGCGCCGCTGCTGCC3' (SEQ ID NO:31) (BbsI
recognition is underlined), leaving the amino acid
sequence-SSGAGQKR- at the 3' end with a Bbs1 site leaving an
overhang at the -KR- dibasic cleavage sequence. The coding region
of .alpha.-factor will be amplified from Cadus 1219 with a Bbs1
site at the 5' end and a BglII site at the 3' end using the primers
5'GGGGAAGACCCGCAGGAGGCAGAAGCTT GGTTGCAG3' (SEQ ID NO:32) (BbsI site
is underlined) and 5'GGGAGATCTTCAGTACATTGGTTGGCC3' (SEQ ID NO:33)
(BglII site is underlined, termination codon is bold). The PCR
fragment encoding the pre-pro segment of POMC is restricted with
HindIII and BbsI and gel purifed, the PCR fragment
encoding_.alpha.-factor is cut with BbsI and BglII and gel
purified, and Cadus 1215 is cut with BglII and partially with
HindIII and the HindIII-BglII restricted vector containing the
pAlter polylinker sequences is gel purified. Three-part ligation of
the two PCR products with HindIII and BglII digested Cadus 1215
will yield a hybrid POMC/.alpha.-factor gene in which the first 104
amino acids residues are from POMC and the remaining 17 are from
.alpha.-factor. The structure of this hybrid gene around the PC1
cleavage site is: --RNSSSSGSSGAGOKREAEAWfHWLQLKPGQPMY* (SEQ ID
NO:34) where residues donated by POMC are underlined, the dibasic
cleavage site is underlined bold, and the sequence of mature
.alpha.-factor is in italics. The tetrapeptide -EAEA- juxtaposed
between the dibasic cleavage site and the amino-terminal tryptophan
of mature .alpha.-factor should be removed by the dipeptidyl
aminopeptidase activity of ste13p.
[0443] Introduction of the PC1-encoding plasmid and the
POMC/.alpha.-factor plasmid into a kex2.DELTA._ strain with the
genotype MATa bar1::hisG far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1
leu2 his3 should result in autocrine growth that is dependent on
PC1-mediated cleavage of the dibasic motif at the amino-terminal
side of the single copy of .alpha.-factor encoded by the
POMC/.alpha.-factor plasmid. That is, the autocrine behaviour of
this strain should be due to expression of PC1. A suitable negative
control is provided by expression of mammalian PC2 in place of PC1;
the cleavage site of POMC that is inserted upstream of the
.alpha.-factor gene is not recognized by PC2. Therefore, it should
be possible to construct strains specific for PC1- versus
PC2-dependent processing by judicious choice of cleavage sites from
POMC to append to the 5' end of the .alpha.-factor gene. Compounds
or random peptides that disrupt the autocrine growth of this strain
but which do not have growth inhibitory effects when this strain is
grown in histidine-containing media are potential inhibitors of PC1
activity.
EXAMPLE 9
Prophetic Example of Substitution of Human MEK (MAP Kinase Kinase)
for Yeast STE7
[0444] In order to develop a screen for compounds which act as
inhibitors of a mammalian kinase, one could construct a yeast
strain that is deficient in STE7 activity, and which contains human
MEK in a yeast expression vector. In addition, the strain would be
equipped with reporter capacities, as described below.
[0445] To disrupt the yeast STE7 gene, the following approach could
be taken: pBluescriptKS+, a multicopy E. coli vector, is engineered
to contain STE7 sequences deleted for 5' noncoding and promoter
sequence and for a sizeable portion of the coding region. The
.vertline.ste7 sequence is then subcloned into
pRS406.vertline.ClaI, a Bluescript-based plasmid containing the
yeast URA3 gene and the resulting plasmid,
pRS406.vertline.ClaI.vertline.ste7, is used to disrupt the wild
type STE7 gene as follows. pRS406.vertline.ste7 is digested with
ClaI, and used to transform yeast strain CY252 (genotype MATa
ste14::TRP1 fus1-HIS3 far1-1 ura3 leu2 trp1 his3 ade2-1 met1) to
uracil prototrophy. Subsequent transfer of Ura+ transformants to
media containing 5-fluoroorotic acid results in colonies containing
the ste7.vertline. allele, which is confirmed by Southern analysis
and by the inability of the strain to grow in the absence of
histidine when stimulated with .alpha. pheromone.
[0446] To construct a plasmid capable of expressing human MEK in
yeast cells, the following oligonucleotides are constructed:
5'-
25 5'-CCGCGTCTCACATGCCCAAGAAGAAGCCG-3' (SEQ ID NO:35) (forward) and
5'-CCGTCTAGATGCTGGCAGCGTGGG-3' (SEQ ID NO:36) (reverse).
[0447] (reverse). When used in a polymerase chain reaction (PCR)
with human cDNA as template, these primers will direct the
amplification of DNA encoding human MEK. To insert the human MEK
gene into a yeast expression vector, the PCR product is digested
with Esp3I and XbaI (bold sequences above) and ligated to the
yeast-E. coli shuttle plasmid Cadus1289, previously digested with
NcoI and XbaI. The resulting plasmid will replicate autonomously in
yeast cells, confer leucine prototrophy to a yeast leu2 mutant, and
direct the expression of MEK from the constitutive PGK1
promoter.
[0448] When the above-described PGK1-MEK plasmid is introduced into
the CY252-ste7.vertline. cells, it should restore their ability to
grow in the absence of histidine when incubated with .alpha.
pheromone, due to the ability of MEK to functionally replace STE7
in the pheromone response pathway and thereby effect the
stimulation of the fus1-HIS3 fusion situated in the chromosome. One
could then screen for compounds which are able to reverse .alpha.
pheromone-dependent growth in the absence of histidine, but which
have no nonspecific toxic effect in the presence of histidine.
[0449] In an autocrine embodiment, the yeast cells made
ste7.vertline. are of the genotype MATa bar1::hisG far1-1 fus1-HIS3
ste14::trp1::LYS2 ura3 trp1 leu2 his3 lys2 (CY588trp). The
procedure followed is exactly as above for CY252. After
construction of CY588trpste7.vertline., the cells created are
transformed with the plasmid expressing MEK, as well as with a
plasmid capable of expressing secreted .alpha. pheromone. This
transformed strain (CY588trpste7.vertline. [MEK/MF.alpha.]) should
be able to grow on media lacking histidine and containing high (20
22222222222222 mM) amounts of 3-aminotriazole. The growth of this
strain on this media should be strictly dependent on the presence
of both plasmids (each necessary, neither sufficient). Compounds
that interfere with this growth could be tested as above, with the
exception that exogenously added .alpha. pheromone is
unnecessary.
[0450] In addition, CY588trpste7.vertline. [MEK/MF.alpha.] could be
transformed with a plasmid library expressing cytoplasmically
targeted random peptides. Those that interfere with the function of
MEK would be identified by replica plating to media deficient in
histidine (and potentially containing 3-aminotriazole); cells
expressing such inhibitory peptides would be His.sup.-. Such a
screen could be streamlined with the addition of reporter
constructs with the potential of negative selection, such as
fus1-URA3 or fus1-GAL1. In this case inhibitory peptides would
confer on the target strain the ability to grow on 5-FOA- (for
cells with diminished fus1-URA3) or galactose- (for cells with
diminished fus1-GAL1 in a gal10.sup.- background) containing
media.
[0451] Confirmatory tests would include biochemical assay of the
activity of MEK (either in vitro or in vivo) in the presence of
random peptides or other molecules identified as potential MEK
inhibitors.
EXAMPLE 10
Functional Expression of a Mammalian G Protein-Coupled Receptor and
Ligand on an Autocrine Yeast Strain
[0452] In this example we describe a set of experiments that detail
the accomplishment of the following: (1) expression of human C5a
receptor in yeast; (2) expression of the native ligand of this
receptor, human C5a, in yeast; and (3) activation of the endogenous
yeast pheromone pathway upon stimulation of the C5a receptor by C5a
when both of these molecules are expressed within the same strain
of autocrine yeast. Following the experimental data we outline the
utility of autocrine strains of yeast that functionally express the
human C5a receptor.
[0453] Human C5a is a 74 amino acid polypeptide that derives from
the fifth component of complement during activation of the
complement cascade; it is the most potent of the complement-derived
anaphylatoxins. C5a is a powerful activator of neutrophil and
macrophage functions including production of cytotoxic superoxide
radicals and induction of chemotaxis and adhesiveness. In addition
C5a stimulates smooth muscle contraction, induces degranulation of
mast cells, induces serotonin release from platelets and increases
vascular permeability. The C5a anaphylatoxin can also amplify the
inflammatory response by stimulating the production of cytokines.
As C5a is a highly potent inflammatory agent, it is a primary
target for the development of antagonists to be used for
intervention in a variety of inflammatory processes.
[0454] The C5a receptor is present on neutrophils, mast cells,
macrophages and smooth muscle cells and couples through G proteins
to transmit signals initiated through the binding of C5a.
[0455] Expression of the C5a Receptor
[0456] The plasmid pCDM8-C5aRc, bearing cDNA sequence encoding the
human C5a receptor, was obtained from N. Gerard and C. Gerard
(Harvard Medical School, Boston, Mass.) (Gerard and Gerard 1991).
Sequence encoding C5a was derived from this plasmid by PCR using
VENT polymerase (New England Biolabs Inc., Beverly Mass.), and the
following primers:
26 #1 - GGTGGGAGGGTGCTCTCTAGAAGGAAGTGTTCACC (SEQ ID NO:62) #2 -
GCCCAGGAGACCAGACCATGGACTCCTTCAATATACCACC. (SEQ ID NO:63)
[0457] Primer #1 contains a single base-pair mismatch (underlined)
to C5a receptor cDNA. It introduces an XbaI site (in bold) 201 bp
downstream from the TAG termination codon of the C5a receptor
coding sequence. Primer #2 contains two mismatched bases and serves
to create an NcoI site (in bold) surrounding the ATG initiator
codon (double underlined). The second amino acid is changed from an
aspartic acid to an asparagine residue. This is the only change in
primary amino acid sequence from the wild type human C5a
receptor.
[0458] The PCR product was restricted with NcoI and XbaI (sites in
bold) and cloned into CADUS 1002 (YEp51Nco), a Gal10 promoter
expression vector. The sequence of the entire insert was determined
by dideoxy sequencing using multiple primers. The sequence between
the NcoI and XbaI sites was found to be identical to the human C5a
receptor sequence that was deposited in GenBank (accession# J05327)
with the exception of those changes encoded by the PCR primers. The
C5a receptor-encoding insert was transferred to CADUS 1289 (pLPXt),
a PGK promoter expression vector, using the NcoI and XbaI sites, to
generate the C5a receptor yeast expression clone, CADUS 1303.
[0459] A version of the C5a receptor which contains a yeast
invertase signal sequence and a myc epitope tag at its amino
terminus was expressed in Cadus 1270-transferred yeast under
control of a GAL10 promoter. Plasmids encoding an untagged version
of the C5a receptor and a myc-tagged derivative of FUS1 served as
controls. The expression of the tagged receptor in yeast was
confirmed by Western blot using the anti-myc monoclonal antibody
9E10. In the lane containing the extract from the Cadus
1270-transformant, the protein that is reactive with the anti-myc
monoclonal antibody 9E10 was approximately 40 kD in size, as
expected. Note that this receptor construct is not identical to the
one used in the autocrine activation experiments. That receptor is
not tagged, does not contain a signal sequence and is driven by the
PGK promoter.
[0460] Expression of the Ligand, C5a
[0461] A synthetic construct of the sequence encoding C5a was
obtained from C. Gerard (Harvard Medical School, Boston, Mass.).
This synthetic gene had been designed as a FLAG-tagged molecule for
secretion from E. coli (Gerard and Gerard 1990). The C5a coding
region, still containing E. coli codon bias, was amplified using
VENT polymerase (New England Biolabs Inc., Beverly Mass.) through
30 cycles using the following primers:
27 C5a5' = CCCCTTAAGCGTGAGGCAGAAGCTACTCTGCAAAAGAAGATC (SEQ ID
NO:64) and C5a3' = GAAGATCTTCAGCGGCCGAGTTGCATGTC (SEQ ID NO:65)
[0462] A PCR product of 257 bp was gel isolated, restricted with
AflII and BglII, and cloned into CADUS 1215 (an expression vector
designed to express peptide sequences in the context of Mf.alpha.)
to yield CADUS 1297. The regions of homology to the synthetic C5a
gene are underlined. The 5' primer also contains pre-pro
.alpha.-factor sequence. Upon translation and processing of the
pre-pro .alpha.-factor sequence, authentic human C5a should be
secreted by yeast containing CADUS 1297. The insert sequence in
CADUS 1297 was sequenced in both orientations by the dideoxy method
and found to be identical to that predicted by the PCR primers and
the published sequence of the synthetic C5a gene (Franke et al.
1988).
[0463] Two sets of experiments, aside from the autocrine activation
of yeast detailed below, demonstrated that CADUS 1297 can be used
to express C5a in yeast.
[0464] 1.) C5a was immunologically detected in both culture
supernatant and lysed cells using a commercially available
enzyme-linked immunosorbent assay (ELISA) (Table 3). This assay
indicated the concentration of C5a in the culture supernate to be
approximately 50 to 100 nM. In comparison, in data derived from
mammalian cells, the binding constant of C5a to its receptor is 1
nM (Boulay et al. 1991).
[0465] 2.) C5a expressed in yeast was shown to compete for binding
with commercially obtained (Amersham Corporation, Arlington
Heights, Ill.), radiolabeled C5a on induced HL60 cells.
[0466] Activation of the Pheromone Response Pathway in Autocrine
Yeast Expressing the Human C5a Receptor and Human C5a
[0467] Activation of the yeast pheromone response pathway through
the interaction of C5a with the C5a receptor was demonstrated using
a growth read-out. The strain used for this analysis, CY455
(MAT.alpha. tbt1-1 ura3 leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1
ste3*1156), contains the following significant modifications. A
pheromone inducible HIS3 gene, fus1-HIS3, is integrated at the FUS1
locus. A hybrid gene containing sequence encoding the first 41
amino acids of GPA1 (the yeast G.alpha. subunit) fused to sequence
encoding human G.alpha.i2a (minus codons for the N-terminal 33
amino acids) replaces GPA1 at its normal chromosomal location. The
yeast STE14 gene is disrupted to lower the basal level of signaling
through the pheromone response pathway. The yeast a-factor receptor
gene, STE3, is deleted. The last two modifications are probably not
essential, but appear to improve the signal-to-noise ratio.
[0468] CY455 (MAT.alpha. tbt1-1 ura3 leu2 trp1 his3 fus1-HIS3 can1
ste14::TRP1 ste3*1156) was transformed with the following
plasmids:
28 Cadus 1289 + Cadus 1215 = Receptor.sup.- Ligand.sup.- = (R-L-)
Cadus 1303 + Cadus 1215 = Receptor.sup.+ Ligand.sup.- = (R+L-)
Cadus 1289 + Cadus 1297 = Receptor.sup.- Ligand.sup.+ = (R-L+)
Cadus 1303 + Cadus 1297 = Receptor.sup.+ Ligand.sup.+ = (R+L+)
Receptor refers to the human C5a receptor. Ligand refers to human
C5a.
[0469] Three colonies were picked from each transformation and
grown overnight in media lacking leucine and uracil, at pH 6.8 with
25 mM PIPES (LEU.sup.- URA.sup.- pH 6.8 with 25 mM PIPES). This
media was made by adding 0.45 ml of sterile 1 M KOH and 2.5 ml of
sterile 1 M PIPES pH 6.8 to 100 ml of standard SD LEU.sup.- URA7
media. After overnight growth the pH of this media is usually
acidified to approximately pH 5.5. Overnight cultures were washed
once with 25 mM PIPES pH 6.8 and resuspended in an equal volume of
media lacking leucine, uracil and histidine (LEU.sup.- URA.sup.-
HIS.sup.- pH6.8 with 25 mM PIPES). The optical density at 600 nm of
a 1/20 dilution of these cultures was determined and the cultures
were diluted into 25 mM PIPES pH 6.8 to a final OD.sub.600 of 0.2.
A volume (5 .mu.l) of this dilution equivalent to 10,000 cells was
spotted onto selective (HIS.sup.- TRP.sup.- pH6.8+1 mM
aminotriazole) or non-selective (HIS.sup.+ TRP.sup.- pH6.8) plates.
Only those strains expressing both C5a and its receptor (R+L+) show
growth on the selective plates which lack histidine. All test
strains are capable of growth on plates containing histidine. The
R+L+ strain will grow on plates containing up to 5 mM
aminotriazole, the highest concentration tested.
[0470] For verification of pheromone pathway activation and
quantification of the stimulation, the activity of the fusl
promoter was determined colorometrically using a fus1-lacZ fusion
in a similar set of strains. CY878 (MAT.alpha. tbt1-1 fus1-HIS3
can1 ste14::trp1::LYS2 ste3*1156 gpa1 (41)-G.alpha.i2) was used as
the starting strain for these experiments. This strain is a trp1
derivative of CY455. The transformants for this experiment
contained CADUS 1584 (pRS424-fus1-lacZ) in addition to the receptor
and ligand plasmids. Four strains were grown overnight in SD
LEU.sup.- URA.sup.- TRP.sup.- pH6.8 with 50 mM PIPES to an
OD.sub.600 of less than 0.8. Assay of .beta.-galactosidase activity
(Guarente 1983) in these strains yields the data shown in FIG.
10.
[0471] Projected Uses of the Autocrine C5a Strains
[0472] A primary use of the autocrine C5a strains will be in the
discovery of C5a antagonists. Inhibitors of the biological function
of C5a would be expected to protect against tissue damage resulting
from inflammation in a wide variety of inflammatory disease
processes including but not limited to: respiratory distress
syndrome (Duchateau et al. 1984; Hammerschmidt et al. 1980), septic
lung injury (Olson et al. 1985), arthritis (Banerjee et al. 1989),
ischemic and post-ischemic myocardial injury (Weisman 1990;
Crawford et al. 1988) and burn injury (Gelfand et al. 1982).
[0473] The autocrine C5a system as described can be used to isolate
C5a antagonists as follows:
[0474] 1. High Throughput Screens to Identify Antagonists of
C5a
[0475] A straightforward approach involves screening compounds to
identify those which inhibit growth of the R+L+ strain described
above in selective media but which do not inhibit the growth of the
same strain or of a R+L- strain in non-selective media. The
counterscreen is necessary to eliminate from consideration those
compounds which are generally toxic to yeast. Initial experiments
of this type have led to the identification of compounds with
potential therapeutic utility.
[0476] 2. Identification of Antagonists Using Negative
Selection
[0477] Replacement of the fus1-HIS3 read-out with one of several
negative selection schemes (fus1-URA3/FOA, fus1-GAL1/galactose or
deoxygalactose, FAR1 sst2 or other mutations that render yeast
supersensitive for growth arrest) would generate a test system in
which the presence of an antagonist would result in the growth of
the assay strain. Such an approach would be applicable to
high-throughput screening of compounds as well as to the selection
of antagonists from random peptide libraries expressed in autocrine
yeast. Optimization of screens of this type would involve screening
the R+L+ strain at a concentration of amino-triazole which ablates
growth of the R+L- strain (we are currently using 0.6 to 0.8 mM)
and counterscreening the R+L- strain at a concentration of
aminotriazole which gives an identical growth rate (we are using
0.14 mM). In addition, the system could employ one of several
colorometric, fluorescent or chemiluminescent readouts. Some of the
genes which can be fused to the fus1 promoter for these alternate
read-outs include lacZ (colorometric and fluorescent substrates),
glucuronidase (colorometric and fluorescent substrates),
phosphatases (e.g. PHO3, PHO5, alkaline phosphatase; colorometric
and chemiluminescent substrates), green protein (endogenous
fluorescence), horse radish peroxidase (colorometric), luciferase
(chemiluminescence).
[0478] The autocrine C5a strains have further utility as
follows:
[0479] 3. In the Identification of Novel C5a Agonists from Random
Peptide Libraries Expressed in Autocrine Yeast
[0480] Novel peptide agonists would contribute to
structure/function analyses used to guide the rational design of
C5a antagonists.
[0481] 4. In the Identification of Receptor Mutants
[0482] Constitutively active, that is, ligand independent,
receptors may be selected from highly mutagenized populations by
growth on selective media. These constitutively active receptors
may have utility in permitting the mapping of the sites of
interaction between the receptor and the G-protein. Identification
of those sites may be important to the rational design of drugs to
block that interaction. In addition, receptors could be selected
for an ability to be stimulated by some agonists but not others or
to be resistant to antagonist. These variant receptors would aid in
mapping sites of interaction between receptor and agonist or
antagonist and would therefore contribute to rational drug design
efforts.
[0483] 5. In the Identification of Molecules That Interact With
G.alpha.i2
[0484] Compounds or peptides which directly inhibit GDP exchange
from G.alpha.i2 would have the same effect as C5a antagonists in
these assays. Additional information would distinguish inhibitors
of GDP exchange from C5a antagonists. This information could be
obtained through assays that determine the following:
[0485] 1. inhibition by test compounds of G.alpha.i2 activation
from other receptors,
[0486] 2. failure of test compounds to compete with radiolabeled
C5a for binding to the C5a receptor,
[0487] 3. failure of test compounds to inhibit the activation of
other G.alpha. subunits by C5a, and
[0488] 4. inhibition by test compounds of signalling from
constitutively active versions of C5a, or other, receptors.
EXAMPLE 11
Construction of Hybrid G.alpha. Genes Construction of Two Sets of
Chimeric Yeast/Mammalian G.alpha. Genes, GPA.sub.41-G.alpha. and
GPA1.sub.Bam-G.alpha.
[0489] The G.alpha. subunit of heterotrimeric G proteins must
interact with both the .beta..gamma. complex and the receptor.
Since the domains of G.alpha. required for each of these
interactions have not been completely defined and since our final
goal requires G.alpha. proteins that communicate with a mammalian
receptor on one hand and the yeast .beta..gamma. subunits on the
other, we desired to derive human-yeast chimeric G.alpha. proteins
with an optimized ability to perform both functions. From the
studies reported here we determined that inclusion of only a small
portion of the amino terminus of yeast G.alpha. is required to
couple a mammalian G.alpha. protein to the yeast .beta..gamma.
subunits. It was anticipated that a further benefit to using these
limited chimeras was the preservation of the entire mammalian
domain of the G.alpha. protein believed to be involved in receptor
contact and interaction. Thus the likelihood that these chimeras
would retain their ability to interact functionally with a
mammalian receptor expressed in the same yeast cell was expected to
be quite high.
[0490] Plasmid Constructions
[0491] pRS416-GPA1 (Cadus 1069). An XbaI-SacI fragment encoding the
entire GPA1 promotor region, coding region and approximately 250
nucleotides of 3' untranslated region was excised from
YCplac111-GPA1 (from S. Reed, Scripps Institute) and cloned into
YEp vector pRS416 (Sikorski and Hieter, Genetics 122: 19 (1989))
cut with XbaI and SacI.
[0492] Site-directed mutagenesis of GPA1 (Cadus 1075, 1121 and
1122). A 1.9 kb EcoRI fragment containing the entire GPA1 coding
region and 200 nucleotides from the 5' untranslated region was
cloned into EcoRI cut, phosphatase-treated pALTER-1 (Promega) and
transformed by electroporation (Biorad Gene Pulser) into
DH5.alpha.F' bacteria to yield Cadus 1075. Recombinant phagemids
were rescued with M13KO7 helper phage and single stranded
recombinant DNA was extracted and purified according to the
manufacturer's specifications. A new NcoI site was introduced at
the initiator methionine of GPA1 by oligonucleotide directed
mutagenesis using the synthetic oligonucleotide:
5'GATATATTAAGGTAGGAAACCATGGGGTGTACAG- TGAG 3'. Positive clones were
selected in ampicillin and several independent clones were
sequenced in both directions across the new NcoI site at +1. Two
clones containing the correct sequences were retained as Cadus 1121
and 1122.
[0493] Construction of a GPA1-based expression vector (Cadus 1127).
The vector used for expression of full length and hybrid mammalian
G.alpha. proteins in yeast, Cadus 1127, was constructed in the
following manner. A 350 nucleotide fragment spanning the 31'
untranslated region of GPA1 was amplified with Taq polymerase
(AmpliTaq Perkin Elmer) using the oligonucleotide primers A
(5'CGAGC-GCTCGAGGGAACGTATAATTAAAQTAGTG, 3') and B
(5'GCGCGGTACCAAGCTTC-AATTCGAGATAATACCC 3'). The 350 nucleotide
product was purified by gel electrophoresis using GeneClean II
(Bio101) and was cloned directly into the pCRII vector by single
nucleotide overlap TA cloning (InVitrogen). Recombinant clones were
characterized by restriction enzyme mapping and by
dideoxynucleotide sequencing. Recombinant clones contained a novel
XhoI site 5' to the authentic GPA1 sequence and a novel KpnI site
3' to the authentic GPA1 sequence donated respectively by primer A
and primer B.
[0494] The NotI and SacI sites in the polylinker of Cadus 1013
(pRS414) were removed by restriction with these enzymes followed by
filling in with the Klenow fragment of DNA polymerase I and blunt
end ligation to yield Cadus 1092. The 1.4 kb PstI-EcoRI 5' fragment
of GPA1 from YCplac111-GPA1 containing the GPA1 promoter and 5'
untranslated region of GPA1 was purified by gel electrophoresis
using GeneClean (Bio101) and cloned into PstI-EcoRI restricted
Cadus 1013 to yield Cadus 1087. The PCR amplified XhoI-KpnI
fragment encoding the 3' untranslated region of GPA1 was excised
from Cadus 1089 and cloned into XhoI-KpnI restricted Cadus 1087 to
yield Cadus 1092. The Not1 and Sac1 sites in the polylinker of
Cadus 1092 were removed by restriction with these enzymes, filling
in with the Klenow fragment of DNA polymerase I, and blunt end
ligation to yield Cadus 1110. The region of Cadus 1122 encoding the
region of GPA1 from the EcoRI site at -200 to +120 was amplified
with Vent DNA polymerase (New England Biolabs, Beverly, Mass.) with
the primers
29 5' CCCGAATCCACCAATTTCTTTACG 3' and 5'
GCGGCGTCGACGCGGCCGCGTAACAGT 3'.
[0495] The amplified product, bearing an EcoRI site at its 5' end
and novel SacI, NotI and SalI sites at its 3' end was restricted
with EcoRI and SalI, gel purified using GeneClean II (Bio101), and
cloned into EcoRI and SalI restricted Cadus 1110 to yield Cadus
1127. The DNA sequence of the vector between the EcoRI site at -200
and the KpnI site at the 3' end of the 3' untranslated region was
verified by restriction enzyme mapping and dideoxynucleotide DNA
sequence analysis.
[0496] PCR amplification of GPA.sub.41-G.alpha. proteins and
cloning into Cadus 1127. cDNA clones encoding the human G alpha
subunits G.alpha.s,_G.alpha.i2, G.alpha.i3, and S. cerevisiae GPA1
were amplified with Vent thermostable polymerase (New England
Bioloabs, Beverly, Mass.). The primer pairs used in the
amplification are as follows:
30 G.alpha.S Primer 1: 5'CTGCTGGAGCTCCGCCTGCTGCTGCTGGGTGCTGGAG3'
(SacI 5') Primer 2: 5'CTGCTGGTCGACGCGGCCGCGGGGGTTCCTTCTTAGAAGCAG-
C3' (SalI 3') Primer 3: 5'GGGCTCGAGCCTTCTTAGAGCAGCTCGTAC3' (XhoI
3') G.alpha.i2 Primer 1: 5'CTGCTGGAGCTCAAGTTGCTGCTGTTGGGT-
GCTGGGG3' (SacI 5') Primer 2: 5'CTGCTGGTCGACGCGGCCGCGCCCCTCAGAAGAG-
GCCGCGGTCC3' (SalI 3') Primer 3: 5'GGGCTCGAGCCTCAGAAGAGGCCGCAGTC3'
(XhoI 3') G.alpha.i3 Primer 1: 5'CTGCTGGAGCTCAAGCTGCTGCTA-
CTCGGTGCTGGAG3' (SacI 5') Primer 2: 5'CTGCTGGTCGACGCGGCCGCCACTAACA-
TCCATGCTTCTCAATAAAGTC3' (SalI 3')
[0497] Primer 3: 5'GGGCTCGAGCATGCTTCTCAATAAAGTCCAC3' (XhoI 3')
After amplification, products were purified by gel electrophoresis
using GeneClean II (Bio101) and were cleaved with the appropriate
restriction enzymes for cloning into Cadus 1127.
[0498] The hybrid GPA.sub.41-G.sub..alpha. subunits were cloned via
a SacI site introduced at the desired position near the 5' end of
the amplified genes and a SalI or XhoI site introduced in the 3'
untranslated region. Ligation mixtures were electroporated into
competent bacteria and plasmid DNA was prepared from 50 cultures of
ampicillin resistant bacteria.
[0499] Construction of Integrating Vectors Encoding
GPA.sub.41-G.sub..alpha. Subunits. The coding region of each
GPA.sub.41-G.alpha. hybrid was cloned into an integrating vector
(pRS406=URA3 AmpR) using the BssHII sites flanking the polylinker
cloning sites in this plasmid. Cadus 1011 (pRS406) was restricted
with BssHII, treated with shrimp alkaline phosphatase as per the
manufacturer's specifications, and the linearized vector was
purified by gel electrophoresis. Inserts from each of the
GPA.sub.41-G.alpha. hybrids were excised with BssHII from the
parental plasmid, and subcloned into gel purified Cadus 1011.
[0500] Construction of GPA.sub.Bam-G.alpha. Constructs. A novel
BamHI site was introduced in frame into the GPA1 coding region by
PCR amplification using Cadus 1179 (encoding a wildtype GPA1 allele
with a novel NcoI site at the initiator methionine) as the
template, VENT polymerase, and the following primers: Primer
A=5'GCATCCATCAATAATCCAG3' and Primer B=5' GAAACAATGGA TCCACTTCTTAC
3'. The 1.1 kb PCR product was gel purified with GeneClean II
(Bio101), restricted with NcoI and BamHI and cloned into NcoI-BamHI
cut and phosphatased Cadus 1122 to yield Cadus 1605. The sequence
of Cadus 1605 was verified by restriction analysis and
dideoxy-sequencing of double-stranded templates. Recombinant
GPA.sub.Bam-G.alpha._hybrids of G.alpha.s, G.alpha.i2, and
G.alpha.16 were generated. Construction of Cadus 1855 encoding
recombinant GPA.sub.Bam-G.alpha..sub.--16 serves as a master
example: construction of the other hybrids followed an analogous
cloning strategy. The parental plasmid Cadus 1617, encoding native
G.alpha.16, was restricted with NcoI and BamHI, treated with shrimp
alkaline phosphatase as per the manufacturer's specifications and
the linearized vector was purified by gel electrophoresis. Cadus
1605 was restricted with NcoI and BamHI and the 1.1 kb fragment
encoding the amino terminal 60% of GPA1 with a novel BamHI site at
the 3' end was cloned into the NcoI- and BamHI-restricted Cadus
1617. The resulting plasmid encoding the
GPA.sub.Bam-G.alpha..sub.-- -16 hybrid was verified by restriction
analysis and assayed in tester strains for an ability to couple to
yeast G.beta..gamma. and thereby suppress the gpa1 null phenotype.
Two additional GPA.sub.Bam-G.alpha._hyb- rids,
GPA.sub.Bam-G.alpha.s_ and GPA.sub.Bam-G.alpha.i2, described in
this application were prepared in an analogous manner using
Cadus1606 as the parental plasmid for the construction of the
GPA.sub.Bam-G.alpha._i2 hybrid and Cadus 1181 as the parental
plasmid for the construction of the GPA.sub.Bam-G.alpha._s
hybrid.
[0501] Coupling by chimeric G.alpha._proteins._The G.alpha.
chimeras described above were tested for the ability to couple a
mammalian G protein-coupled receptor to the pheromone response
pathway in yeast. The results of these experiments are outlined in
Table 5. Results obtained using GPA1.sub.41-G.alpha.i2 to couple
the human C5a receptor to the pheromone response pathway in
autocrine strains of yeast are disclosed in Example 10 above.
31TABLE 1 ABC TRANSPORTERS* Species System Substrate Bacteria
Salmonella OppABCDF Oligopeptides typhimurium Streptococcus
AmiABCDEF Oligopeptides pneumoniae Bacillus Opp (SpoOK)
Oligopeptides subtilis E. coli Dpp Dipeptides Bacilus subtilis DciA
Dipeptides S. typhimurium HisJQMP Histidine E. coli HisJQMP
Histidine E. coli MalEFGK Maltose S. typhimurium MalEFGK Maltose
Enterobacter MalEFGK Maltose aerogenes E. coli UgpABCE
sn-Glycerol-3- phosphate E. coli AraFGH Arabinose E. coli RbsACD
Ribose E. coli GlnHPQ Glutamine S. typhimurium ProU (VWX)
Glycine-betaine E. coli ProU (VWX) Glycine-betaine E. coli LivHMGF
(JK) Leucine- isoleucine-valine E. coli PstABC Phosphate
Pseudomonas NosDYF Copper stutzeri E. coli ChlJD Molybdenum E. coli
CysPTWAM Sulphate- Thiosulfate E. coli BtuCDE Vitamin B12 E. coli
FhuBCD Fe.sup.3+-ferrichrome E. coli FecBCDE Fe.sup.3+-dicitrate S.
marcescens SfuABC Fe.sup.3+ Mycoplasma p37, 29, 69 ? E. coli
Phn/Psi Alkyl-phosphonates (?) Streptomyces DrrAB
Daunomycin/Doxorubicin peucetius Streptomyces TlrC Tylosin fradiae
Staphylococcus MsrA Erythromycin resistance Agrobacterium OccJQMP
Octopine tumefaciens E. coli HlyB Haemolysin Pasturella LtkB
leukotoxin E. coli CvaB Colicin V Erwinia PrtD Proteases
chrysanthemi Bordetella CyaB Cyclolysin pertussis Streptococcus
ComA Competence factor pneumoniae Rhizobium meliloti NdvA
.beta.-1,2-glucan Agrobacterium ChvA .beta.-1,2-glucan tumefaciens
Haemophilus BexAB Capsule influenzae polysaccharide E. coli KpsMT
Capsule polysaccharide Niesseria CrtCD Capsule polysaccharide E.
coli FtsE Cell division E. coli UvrA DNA repair Rhizobium NodI
Nodulation leguminosarum Rhizobium meliloti OFR1 ? Cyanobacteria
Anabaena HetA Differentiation Synchococcus CysA Sulphate Yeast S.
cerevisiae STE6 a-mating peptide S. cerevisiae ADP1 ? S. cerevisiae
EF-3 Translation Protozoa Plasmodium pfMDR Chloroquine Lieshmania
ltpgpA Methotrexate/heavy metals Insect Drosophila white-brown Eye
pigments Drosophila Mdr49 Hydrophobic drugs? Mdr65 ? Plants
Liverwort MbpX ? chloroplast Animals Man CFTR Chloride Mouse CFTR
Chloride Xenopus CFTR Chloride Cow CFTR Chloride Dogfish CFTR
Chloride Man MDR1 Hydrophobic drugs MDR3 ? Mouse MDR1 Hydrophobic
drugs MDR2 ? MDR3 Hydrophobic drugs MDR3 Hydrophobic drugs Hamster
Pgp1 Hydrophobic drugs Pgp2 Hydrophobic drugs Pgp3 ? Man PMP70
Polypeptides? Man RING4-11 Peptides PSF1-PSF2 Peptides Mouse
HAM1-HAM2 Peptides Rat Mtp1 Peptides *Adapted from Higgins, C. F.
1992. ABC Transporters: From Microorganizmz to Man. Annu. Rev. Cell
Biol. 8, 67-113.
[0502]
32TABLE 2 HUMAN G PROTEIN-COUPLED SEVEN TRANSMEMBRANE RECEPTORS:
REFERENCES FOR CLONING Receptor Reference .alpha..sub.1A-adrenergic
receptor Bruno et al. (1991) .alpha..sub.1B-adrenergic receptor
Ramarao et al. (1992) .alpha..sub.2-adrenergic receptor Lomasney et
al. (1990) .alpha..sub.2B-adrenergic receptor Weinshank et al.
(1990) .beta..sub.1-adrenergic receptor Frielle et al. (1987)
.beta..sub.2-adrenergic receptor Kobilka et al. (1987)
.beta..sub.3-adrenergic receptor Regan et al. (1988) m.sub.1 AChR,
m2 AChR, m.sub.3 AChR, Bonner et al. (1987) m.sub.4 AChR Peralta et
al. (1987) m5 AChR Bonner et al. (1988) D.sub.1 dopamine Dearry et
al. (1990) Zhou et al. (1990) Sunahara et al. (1990) Weinshank et
al. (1991) D.sub.2 dopamine Grandy et al. (1989) D.sub.3 dopamine
Sokoloff et al. (1990) D.sub.4 dopamine Van Tol et al. (1991)
D.sub.5 dopamine M. Caron (unpub.) Weinshank et al. (1991) A1
adenosine Libert et al. (1992) adenosine A2b Pierce et al. (1992)
5-HT1a Kobilka et al. (1987) Fargin et al. (1988) 5 -HT1b Hamblin
et al. (1992) Mochizuki et al. (1992) 5HT1-like Levy et al. (1992a)
5-HT1d Levy et al. (1992b) 5HT1d-like Hamblin and Metcalf (1991)
5HT1d beta Demchyshyn et al. (1992) substance K (neurokinin A)
Gerard et al. (1990) substance P (NK1) Gerard, et al. (1991);
Takeda et al. (1991) f-Met-Leu-Phe Boulay et al. (1990) Murphy
& McDermott (1991) DeNardin et al. (1992) angiotensin II type 1
Furuta et al. (1992) mas proto-oncogene Young et al. (1986)
endothelin ETA Hayzer et al. (1992) Hosoda et al. (1991) endothelin
ETB Nakamuta et al. (1991) Ogawa et al. (1991) thrombin Vu et al.
(1991) growth hormone-releasing Mayo (1992) hormone (GHRH)
vasoactive intestinal Sreedharan et al. (1991) peptide (VIP)
oxytocin Kimura et al., (1992) somatostatin SSTR1 and Yamada et al.
(1992a) SSTR2 Yamada et al. (1992b) SSTR3 cannabinoid Gerard et al.
(1991) follicle stimulating Minegish et al. (1991) hormone (FSH)
LH/CG Minegish et al. (1990) thyroid stimulating Nagayama et al.
(1989) hormone (TSH) Libert et al. (1989) Misrahi et al. (1990)
thromboxane A2 Hirata et al. (1991) platelet-activating factor Kunz
et al. (1992) (PAF) C5a anaphylatoxin Boulay et al. (1991) Gerard
and Gerard (1991) Interleukin 8 (IL-8) IL- Holmes et al. (1991) 8RA
IL-8RB Murphy and Tiffany (1991) Delta Opioid Evans et al. (1992)
Kappa Opioid Xie et al. (1992) mip-1/RANTES Neote et al. (1993)
IL-8RB Murphy et al., in press Rhodopsin Nathans and Hogness (1984)
Red opsin, Green opsin, Nathans, et al. (1986) Blue opsin
metabotropic glutamate Tanabe et al. (1992) mGluR1-6 histamine H2
Gantz et al. (1991) ATP Julius, David (unpub.) neuropeptide Y
Herzog et al. (1992) Larhammar et al. (1992) amyloid protein
precursor Kang et al. (1987) Mita, et al. (1988) Lemaire et al.
(1989) insulin-like growth factor Kiess et al. (1988) II bradykinin
Hess et al. (1992) gonadotropin-releasing Chi et al. (1993) hormone
cholecystokinin Pisegna et al. (1992) melanocyte stimulating
Chhajlane et al. (1992) hormone receptor Mountjoy et al. (1992)
antidiuretic hormone Birnbaumer et al. (1992) receptor glucagon
receptor Sprecher et al. (1993) adrenocorticotropic Mountjoy et al.
(1992) hormone II
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[0507] Boulay, F., Tardif, M., Brouchon, L., Vignais, P. (1990) The
human N-formylpeptide receptor. Characterization of two cDNA
isolates and evidence for a new subfamily of G protein-coupled
receptors. Biochemistry 29, 11123-11133.
[0508] Bruno, J. F., Whittaker, J., Song, J. F., Berelowitz, M.
(1991) Molecular cloning and sequencing of a cDNA encoding a human
1A adrenergic receptor. Biochem. Biophys. Res. Commun. 179,
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[0509] De-Nardin-E. Radel-S-J. Lewis-N. Genco-R-J. Hammarskjold-M.
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33TABLE 3 Detection of C5a production in yeast by ELISA. R-L- R+L-
R-L+ R+L+ [C5a] in culture n.d. n.d. 0.64 ng/ml 0.5 ng/ml =77 nM
=60 nM [C5a] released n.d. n.d. 0.8 ng/ml 0.6 ng/ml from lysed
cells* =97 nM =73 nM C5a was detected by enzyme-linked
immunosorbent assay (ELISA). Molar concentrations were calculated
using MW=8273 as predicted by C5a sequence. *Determined by
pelleting cells, resuspending cells in the original volume,
breaking yeast with glass beads and assaying the resulting
supernatant. n.d. = not done
[0581]
34TABLE 4 Coupling of the C5a receptor to G.alpha. chimeras in
yeast. Expression Chimera Context Result GPA1.sub.41-G.alpha.i2
single copy, Good signal to noise ratio: integrated, efficient
coupling to yeast GPA1 promoter .beta..gamma..
GPA1.sub.41-G.alpha.i3 single copy, Poor signal to noise ratio:
integrated, high background due to poor GPA1 promoter coupling to
yeast .beta..gamma., high LIRMA*. GPA1.beta.am-G.alpha.i2 low copy
plasmid, Signal equal to that with GPA 1 promoter
GPA1.sub.14-G.alpha.i2, however, back- ground is greater.
GPA1.beta.am-G.alpha.16 low copy plasmid, Poor signal to noise
ratio, GPA1 promoter high background due to poor coupling to yeast
.beta..gamma., high LIRMA*. GPA1.beta.am-G.alpha.s low copy
plasmid, Unacceptably high background GPA1 promoter due to poor
coupling to yeast .beta.`, high LIRMA*. *LIRMA = Ligand Independent
Receptor Mediated Activation. With this phenomenon, there is an
increase in growth on selective media for strains containing
heterologous receptor in the # absenceof ligand. It is possible
that some receptor antagonists would decrease LIRMA. It has been
noted (Milano, et al. 1994) that specific antagonists reduce LIRMA
of the .beta.2 adrenergic receptor when that receptor is
overexpressed in transgenic mice. LIRMA may be exploited in several
ways, including the identification of antagonists capable of
reducing the phenomenon. A subset of antagonists would be expected
to affect the receptor conformation in such a way as to prevent the
downstream signalling that occurs in the absence of agonist. LIRMA
can be exploited to identify new G protein-coupled receptors by
expressing cDNA clones in yeast strains expressing those chimeric G
proteins which couple only poorly to yeast .beta..gamma.. # In
addition, LIRMA may permit the identification of inhibitors that
are specific for G. proteins.
[0582]
35TABLE 5 Sequence alignments of N-terminal regions of G.alpha.
subunits and N-terminal sequences of GPA.sub.41-G.alpha. hybrid
proteins. A. Alignment of GPA1 with G.alpha. Subunits GPA1
MGC.TVSTQTIGDESDPFLQNKR-
ANDVIEQSLQLEKQRDKNEIKLLLLGAGESGKSTVLKQLKLLHQ..... G.alpha.S
MGCLGTS..KTEDQRNEEKAQREANKKIEKQLQKDKQVYRATHRLLLLGAGESGKSTIVKQMRILHV.....
G.alpha.i2 MGC.TVS........AEDKAAAERSKMIDKNLREDGEKAAREVKLLLL-
GAGESGKSTIVKQMKIIHE..... G.alpha.i3 MGC.TVS........AEDKAAVER-
SKMIDRNLREDGEKAAKEVKLLLLGAGESGKSTIVKQMKIIHE..... G.alpha.16
MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHG.....
B. GPA.sub.41-G.alpha. Hybrids GPA.sub.41- G.alpha.S
MGC.TVSTQTIGDESDPFLQNKRANDVIEQSLQLEKQRDKNERKLLLLGAGESGKSTIVKQMRILHV.....
GPA.sub.41- G.alpha.i2 MGC.TVSTQTIGDESDPFLQNKRANDVIEQSLQLEK-
QRDKNEVKLLLLGAGESGKSTIVKQMKIIHE..... GPA.sub.41- G.alpha.i3
MGC.TVSTQTIGDESDPFLQNKRANDVIEQSLQLEKQRDKNEVKLLLLGAGESGKSTIVKQMKIIHE.....
GPA.sub.41- G.alpha.16 MGC.TVSTQTIGDESDPFLQNKRANDVIEQSLQLEK-
QRDKNELKLLLLGPGESGKSTFIKQMRIIHG.....
[0583]
36TABLE 6 Coupling of G.alpha. switch region hybrids to the
pheromone response pathway. GPA1 amino Gas amino acid Protein acid
sequences sequences Phenotype GPA1 1-472 none Couples with
G.beta..gamma. G.alpha.S none 1-394 Couples with G.beta..gamma.
weakly GPA.sub.41-S 1-41 42-394 Couples with G.beta..gamma. weakly
SGS 297-333 1-201 + 237-394 Does not couple with G.beta..gamma.
GPA.sub.41-SGS 1-41 + 297-333 42-201 + 237-394 Couples with
G.beta..gamma. weakly
[0584]
37TABLE 7 G.alpha. Subunit Alignment - "Switch Region" .beta.2
.beta.3 .alpha.2 .beta.4 GPA.1
RIDTTGITETEFNIGSSKFKVLDAGGQRSERKKWIHCFEGITAVLFVLAMSEYDQMLFEDER
G.alpha.S .VL.S..F..K.QNDKVN.NMF.V....D......Q..NDV..II..V.S.S.NMV-
IR..NQ G.alpha.i2 .VK....V..H.TFKDLH..MF.V................-
V..II.CV.L.A..LV.ADE.M G.alpha.i3 .VK....V..H.TFKDLY..MF.V-
................V..II.CV.L.D..LV.A...E G.alpha.O
.VK....V..H.TFKNLH.RLF.V...............DV..II.CN.L.G...V.H...T
G.alpha.11
.VP....1.YP.DLENII..MV.................NV.SIM.LV.L.....C.- E.NNQ
G.alpha.16 .MP....N.YC.SVQKTNL.IV.................N.-
I.LIYLASL.....V.V.SDN
[0585]
38TABLE 8 G protein-coupled receptors that couple through
G.alpha.i: M2 muscarinic acetylcholine M4 muscarinic acetylcholine
adenosine A1 adenosine A3 .alpha..sub.2A-adrenergic
.alpha..sub.2B-adrenergic .alpha..sub.2C-adrenergic bradykinin
B.sub.2 cannabinoid D2 dopamine D4 dopamine ET.sub.B endothelin
formyl-methionyl peptide receptor FPR.sub.1 metabotropic
glutamateR.sub.2 metabotropic glutamateR.sub.3 metabotropic
glutamateR.sub.4 5HT.sub.1A (serotonin) 5HT.sub.1B (serotonin)
5HT.sub.1D (serotonin) 5HT.sub.1E (serotonin) 5HT.sub.1F
(serotonin) neuropeptide Y delta opioid prostaglandin EP3
somatostatin 2 somatostatin 3 somatostatin 4 thrombin C5a platelet
activating factor angiotensin AT.sub.1 angiotensin AT.sub.2 IL-8
MCP1A MCP1B
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